Accessory functions for use in recombinant AAV virion production

ABSTRACT

Accessory functions capable of supporting efficient recombinant AAV (rAAV) virion production in a suitable host cell are provided. The accessory functions are in the form of one or more vectors that are capable of being transferred between cells. Methods of producing rAAV virions are also provided. The methods can be practiced to produce commercially significant levels of rAAV particles without also generating significant levels of infectious helper virus or other contaminating by-products.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.10/177,871, filed Jun. 19, 2002 which is a divisional of U.S. patentapplication Ser. No. 09/406,363, filed Sep. 28, 1999, which is acontinuation of U.S. patent application Ser. No. 08/745,957, filed Nov.7, 1996, now U.S. Pat. No. 6,004,797, from which applications priorityis claimed under to 35 USC §120, which claims the benefit of provisionalpatent application Ser. No. 60/006,402, filed Nov. 9, 1995, from whichpriority is claimed under 35 USC §119(e)(1) and which applications areincorporated herein by reference in their entireties.

TECHNICAL FIELD

The present invention relates generally to accessory functions for usein adeno-associated virus (AAV) virion production. More particularly,the invention relates to vectors which provide accessory functionscapable of supporting efficient recombinant AAV virion production in asuitable host cell, and methods of use thereof.

BACKGROUND OF THE INVENTION

Gene delivery is a promising method for the treatment of acquired andinherited diseases. A number of viral based systems for gene transferpurposes have been described, such as retroviral systems which arecurrently the most widely used viral vector systems for this purpose.For descriptions of various retroviral systems, see, e.g., U.S. Pat. No.5,219,740; Miller and Rosman (1989) BioTechniques 7:980-990; Miller, A.D. (1990) Human Gene Therapy 1:5-14; Scarpa et al. (1991) Virology180:849-852; Burns et al. (1993) Proc. Natl. Acad. Sci. USA90:8033-8037; and Boris-Lawrie and Temin (1993) Cur. Opin. Genet.Develop. 3:102-109.

Adeno-associated virus (AAV) systems have also been used for genedelivery. AAV is a helper-dependent DNA parvovirus which belongs to thegenus Dependovirus. AAV requires infection with an unrelated helpervirus, either adenovirus, a herpesvirus or vaccinia, in order for aproductive infection to occur. The helper virus supplies accessoryfunctions that are necessary for most steps in AAV replication. In theabsence of such infection, AAV establishes a latent state by insertionof its genome into a host cell chromosome. Subsequent infection by ahelper virus rescues the integrated copy which can then replicate toproduce infectious viral progeny. AAV has a wide host range and is ableto replicate in cells from any species so long as there is also asuccessful infection of such cells with a suitable helper virus. Thus,for example, human AAV will replicate in canine cells co-infected with acanine adenovirus. AAV has not been associated with any human or animaldisease and does not appear to alter the biological properties of thehost cell upon integration. For a review of AAV, see, e.g., Berns andBohenzky (1987) Advances in Virus Research (Academic Press, Inc.)32:243-307.

The AAV genome is composed of a linear, single-stranded DNA moleculewhich contains 4681 bases (Berns and Bohenzky, supra). The genomeincludes inverted terminal repeats (ITRs) at each end which function incis as origins of DNA replication and as packaging signals for thevirus. The ITRs are approximately 145 bp in length. The internalnonrepeated portion of the genome includes two large open readingframes, known as the AAV rep and cap regions, respectively. Theseregions code for the viral proteins involved in replication andpackaging of the virion. In particular, a family of at least four viralproteins are synthesized from the AAV rep region, Rep 78, Rep 68, Rep 52and Rep 40, named according to their apparent molecular weight. The AAVcap region encodes at least three proteins, VP1, VP2 and VP3. For adetailed description of the AAV genome, see, e.g., Muzyczka, N. (1992)Current Topics in Microbiol. and Immunol. 158:97-129.

The construction of recombinant AAV virions has been described. See,e.g., U.S. Pat. Nos. 5,173,414 and 5,139,941; International PublicationNumbers WO 92/01070 (published 23 Jan. 1992) and WO 93/03769 (published4 Mar. 1993); Lebkowski et al. (1988) Molec. Cell. Biol. 8:3988-3996;Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press);Carter, B. J. (1992) Current Opinion in Biotechnology 3:533-539;Muzyczka, N. (1992) Current Topics in Microbiol. and Immunol.158:97-129; and Kotin, R. M. (1994) Human Gene Therapy 5:793-801.

Contemporary recombinant AAV (rAAV) virion production involvesco-transfection of a host cell with an AAV vector plasmid and aconstruct which provides AAV helper functions to complement functionsmissing from the AAV vector plasmid. In this manner, the host cell iscapable of expressing the AAV proteins necessary for AAV replication andpackaging. The host cell is then infected with a helper virus to provideaccessory functions. The helper virus is generally an infectiousadenovirus (type 2 or 5), or herpesvirus.

AAV helper functions can be provided via an AAV helper plasmid thatincludes the AAV rep and/or cap coding regions but which lacks the AAVITRs. Accordingly, the helper plasmid can neither replicate nor packageitself. A number of vectors that contain the rep coding region areknown, including those vectors described in U.S. Pat. No. 5,139,941,having ATCC Accession Numbers 53222, 53223, 53224, 53225 and 53226.Similarly, methods of obtaining vectors containing the HHV-6 homologueof AAV rep are described in Thomson et al. (1994) Virology 204:304-311.A number of vectors containing the cap coding region have also beendescribed, including those vectors described in U.S. Pat. No. 5,139,941.

AAV vector plasmids can be engineered to contain a functionally relevantnucleotide sequence of interest (e.g., a selected gene, antisensenucleic acid molecule, ribozyme, or the like) that is flanked by AAVITRs which provide for AAV replication and packaging functions. After anAAV helper plasmid and an AAV vector plasmid bearing the nucleotidesequence are introduced into the host cell by transient transfection,the transfected cells can be infected with a helper virus, mosttypically an adenovirus, which, among other functions, transactivatesthe AAV promoters present on the helper plasmid that direct thetranscription and translation of AAV rep and cap regions. Uponsubsequent culture of the host cells, rAAV virions (harboring thenucleotide sequence of interest) and helper virus particles areproduced.

When the host cell is harvested and a crude extract is produced, theresulting preparation will contain, among other components,approximately equal numbers of rAAV virion particles and infectioushelper virions. rAAV virion particles can be purified away from most ofthe contaminating helper virus, unassembled viral proteins (from thehelper virus and AAV capsid) and host cell proteins using knowntechniques. Purified rAAV virion preparations that have been producedusing infection with adenovirus type-2 contain high levels ofcontaminants. Particularly, 50% or greater of the total protein obtainedin such rAAV virion preparations is free adenovirus fiber protein.Varying amounts of several unidentified adenoviral and host cellproteins are also present. Additionally, significant levels ofinfectious adenovirus virions are obtained, necessitating heatinactivation. The contaminating infectious adenovirus can be inactivatedby heat treatment (56° C. for 1 hour) and rendered undetectable bysensitive adenovirus growth assays (e.g., by cytopathic effect (CPE) ina permissive cell line). However, heat treatment also results in anapproximately 50 drop in the titer of functional rAAV virions.

Production of rAAV virions using an infectious helper virus (such as anadenovirus type-2, or a herpesvirus) to supply accessory functions isundesirable for several reasons. AAV vector production methods whichemploy a helper virus require the use and manipulation of large amountsof high titer infectious helper virus which presents a number of healthand safety concerns, particularly in regard to the use of a herpesvirus.Also, concomitant production of helper virus particles in rAAV virionproducing cells diverts large amounts of cellular resources away fromrAAV virion production, possibly resulting in lower rAAV virion yields.

More particularly, in methods where infection of cells with adenovirustype-2 are used to provide the accessory functions, more than 95% of thecontaminants found in the purified rAAV virion preparations are derivedfrom adenovirus. The major contaminant, free adenovirus fiber protein,tends to co-purify with rAAV virions on CsCl density gradients due to anon-covalent association between the protein and rAAV virions. Thisassociation makes separation of the two especially difficult, loweringrAAV virion purification efficiency. Such contaminants may beparticularly problematic since many adenoviral proteins, including thefiber protein, have been shown to be cytotoxic (usually at highconcentrations), and thus may adversely affect or kill target cells.Thus, a method of producing rAAV virions without the use of infectioushelper viruses to provide necessary accessory functions would beadvantageous.

A number of researchers have investigated the genetic basis of accessoryfunctions, particularly adenovirus-derived functions. Generally, twoapproaches have been used to attempt to identify those adenoviral genesthat are involved in AAV replication: examination of the ability ofvarious adenovirus mutants to provide accessory functions; and the studyof the effect of transfected adenoviral genes or regions on AAVreplication in the absence of adenovirus infection.

Studies with various adenovirus mutants that are capable of supportingAAV replication (e.g., by supplying necessary accessory functions) at orabout the levels obtained by infection with a wild-type adenovirus havedemonstrated that particular adenovirus genes or gene regions are notinvolved in AAV replication. However, loss-of-function data from suchstudies have failed to provide conclusive information that a particulargene region is involved with AAV replication since many of theadenovirus genes and control regions are overlapping and/or incompletelymapped.

Particularly, adenovirus mutants with fairly well characterizedmutations in the following genes or gene regions have been tested fortheir ability to provide accessory functions necessary for AAV viralreplication: E1a (Laughlin et al. (1982) J. Virol. 41:868, Janik et al.(1981) Proc. Natl. Acad. Sci. USA 78:1925); E1b (Laughlin et al. (1982),supra, Janik et al. (1981), supra, Ostrove et al. (1980) Virology104:502); E2a (Handa et al. (1975) J. Gen. Virol. 29:239, Straus et al.(1976) J. Virol. 17:140, Myers et al. (1980) J. Virol. 35:665, Jay etal. (1981) Proc. Natl. Acad. Sci. USA 78:2927, Myers et al. (1981) J.Biol. Chem. 256:567); E2b (Carter, B. J. (1990) “Adeno-Associated VirusHelper Functions,” in CRC Handbook of Parvoviruses, vol. I (P. Tijssen,ed.); E3 (Carter et al. (1983) Virology 126:505); and E4 (Carter et al.(1983), supra, Carter, B. J. (1995), supra). Poorly characterizedadenovirus mutants that were incapable of DNA replication and late genesynthesis have also been tested (Ito et al. (1970) J. Gen. Virol. 9:243,Ishibashi et al. (1971) Virology 45:317).

Adenovirus mutants with defects in the E2b and E3 regions have beenshown to support AAV replication, indicating that the E2b and E3 regionsare probably not involved in providing accessory functions (Carter etal. (1983), supra). Mutant adenoviruses defective in the E1a region, orhaving a deleted E4 region, are unable to support AAV replication,indicating that the E1a and E4 regions are likely required for AAVreplication, either directly or indirectly (Laughlin et al. (1982),supra, Janik et al. (1981), supra, Carter et al. (1983), supra). Studieswith E1b and E2a mutants have produced conflicting results. Further,adenovirus mutants incapable of DNA replication and late gene synthesishave been shown to be permissive for AAV replication (Ito et al. (1970),supra, Ishibashi et al (1971), supra). These results indicate thatneither adenoviral DNA replication nor adenoviral late genes arerequired for AAV replication.

Transfection studies with selected adenoviral genes have been used in anattempt to establish whether a transfected set of adenovirus genes iscapable of providing the same level of accessory functions for AAVreplication as that provided by an adenovirus infection. Particularly,in vitro AAV replication has been assessed using human 293 cellstransiently transfected with various combinations of adenovirusrestriction fragments encoding single adenovirus genes or groups ofgenes (Janik et al. (1981), supra). Since the above-describedtransfection studies were done in cells that stably express theadenovirus E1a and E1b regions, the requirement for those regions couldnot be tested. However, it was deduced that the combination of threeadenoviral gene regions, VA I RNA, E2a and E4, could provide accessoryfunctions (e.g., support AAV replication) at a level that wassubstantially above background, but that was still approximately 8,000fold below the level provided by infection with adenovirus. When allcombinations of two of the three genes were tested, the accessoryfunction levels ranged between 10,000 to 100,000 fold below the levelsprovided by infection with adenovirus.

Transfection studies with selected herpes simplex virus type-1 (HSV-1)genes have also been conducted in an attempt to establish whether atransfected set of HSV-1 genes is capable of providing the same level ofaccessory functions for AAV replication as that provided by an HSV-1infection. Weindler et al. (1991) J. Virol. 65:2476-2483. However, suchstudies were limited to identifying only those HSV-1 genes necessary tosupport wild-type AAV replication, not rAAV production. Further, theidentified HSV-1 accessory functions were significantly less efficientat supporting AAV replication than adenovirus-derived functions.

Accordingly, there remains a need in the art to identify a subset of theadenovirus genome or functional homologues of the adenovirus genome,that include only those accessory functions required for rAAV virionproduction. The subset can then be included in an accessory functionvector or system which, when introduced into a suitable host cell,supports the production of rAAV virions in an amount that issubstantially equivalent to, or greater than, the amount produced usingan adenovirus infection. Further, there remains a need to provide anaccessory function system that is capable of producing commerciallysignificant levels of rAAV virion particles without also generatingsignificant levels of infectious adenovirus virions, or othercontaminating by-products.

SUMMARY OF THE INVENTION

The present invention is based on the identification of the accessoryfunctions needed to support efficient AAV replication in a suitable hostcell. The invention provides a system which includes such functions andallows for the production of rAAV virions without the use of a helpervirus.

In certain embodiments, the invention relates to nucleic acid moleculesencoding accessory functions and that lack at least one adenoviral lategene region. The molecules can be provided in one or more vectors whichinclude nucleotide sequences derived from an adenovirus type-2 or type-5genome, or functional homologues thereof. Thus, in one aspect, theinvention relates to a vector containing a nucleotide sequence selectedfrom the group consisting of (i) a sequence that provides adenovirus VARNAs, (ii) an adenovirus E4 ORFG coding region, (iii) an adenovirus E2a72 kD coding region (coding for the E2a 72 kD DNA-binding protein), andany combination of nucleotide sequences (i), (ii) and (iii).

In another embodiment, the invention relates to nucleic acid moleculeswhich provide accessory functions capable of supporting efficientrecombinant AAV (rAAV) virion production in a suitable host cell andthat lack at least one adenoviral late gene region and vectorscontaining the nucleic acid molecules.

In another aspect, the invention relates to a vector containing aplurality of nucleotide sequences, including a sequence that provides anadenovirus VA RNA, a sequence comprising an adenovirus E4 coding regionand a sequence comprising an adenovirus E2a coding region.

In yet another aspect of the invention, a vector is provided whichcontains a sequence that provides an adenovirus VA RNA, a sequencecomprising an adenovirus E4 coding region, a sequence comprising anadenovirus E2a coding region, and a sequence comprising an adenovirusE1a and E1b coding region.

In another embodiment of the invention, accessory function systems forrAAV virion production are provided, wherein the systems contain aplurality of accessory function vectors which provide accessory functioncomponents suitable for supporting efficient AAV virion production in asuitable host cell.

In yet another embodiment of the invention, methods for producing rAAVvirions are provided. The methods generally entail (1) introducing anAAV vector into a suitable host cell; (2) introducing an AAV helperconstruct into the cell, wherein the helper. construct is capable ofbeing expressed in the host cell to complement AAV helper functionsmissing from the AAV vector; (3) introducing one or more accessoryfunction vectors into the host cell, wherein the one or more accessoryfunction vectors provide accessory functions capable of supportingefficient rAAV virion production in the host cell; and (4) culturing thecell to produce rAAV virions.

In a further embodiment, recombinant AAV virions produced by the methodsof the present invention are also provided.

These and other embodiments of the subject invention will readily occurto those of ordinary skill in the art in view of the disclosure herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the plasmid construct pladeno 1 which includes VA RNA, E4(containing the ORF 6) and E2a adenoviral gene regions derived fromadenovirus type-5 which were inserted into a pBSII s/k− vector plasmid.

FIG. 2 depicts the plasmid construct pladeno 1 E1 which was formed byinserting a 4,102 bp BsxGI-Eco47III fragment (containing the adenovirustype-5 E1a and E1b coding regions) into the pladeno 1 construct.

FIG. 3 depicts the plasmid construct pladeno 5 which includes the VARNA, E4 ORF 6, and E2a adenoviral gene regions derived from theadenovirus type-2 genome.

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of virology, microbiology, molecularbiology and recombinant DNA techniques within the skill of the art. Suchtechniques are explained fully in the literature. See, e.g., Sambrook,et al. Molecular Cloning: A Laboratory Manual (Current Edition); DNACloning: A Practical Approach, vol. I & II (D. Glover, ed.);Oligonucleotide Synthesis (N. Gait, ed., Current Edition); Nucleic AcidHybridization (B. Hames & S. Higgins, eds., Current Edition);Transcription and Translation (B. Hames & S. Higgins, eds., CurrentEdition); CRC Handbook of Parvoviruses, vol. I & II (P. Tijessen, ed.);Fundamental Virology, 2nd Edition, vol. I & II (B. N. Fields and D. M.Knipe, eds.)

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.

As used in this specification and the appended claims, the singularforms “a,” “an” and “the” include plural references unless the contentclearly dictates otherwise.

A. Definitions

In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

“Gene transfer” or “gene delivery” refers to methods or systems forreliably inserting foreign DNA into host cells. Such methods can resultin transient expression of non-integrated transferred DNA,extrachromosomal replication and expression of transferred replicons(e.g., episomes), or integration of transferred genetic material intothe genomic DNA of host cells. Gene transfer provides a unique approachfor the treatment of acquired and inherited diseases. A number ofsystems have been developed for gene transfer into mammalian cells. See,e.g., U.S. Pat. No. 5,399,346.

By “vector” is meant any genetic element, such as a plasmid, phage,transposon, cosmid, chromosome, virus, virion, etc., which is capable ofreplication when associated with the proper control elements and whichcan transfer gene sequences between cells. Thus, the term includescloning and expression vehicles, as well as viral vectors.

By “adeno-associated virus inverted terminal repeats” or “AAV ITRs” ismeant the art-recognized regions found at each end of the AAV genomewhich function together in cis as origins of DNA replication and aspackaging signals for the virus. AAV ITRs, together with the AAV repcoding region, provide for the efficient excision and rescue from, andintegration of a nucleotide sequence interposed between two flankingITRs into a mammalian cell genome.

The nucleotide sequences of AAV ITR regions are known. See, e.g., Kotin,R. M. (1994) Human Gene Therapy 5:793-801; Berns, K. I. “Parvoviridaeand their Replication” in Fundamental Virology, 2nd Edition, (B. N.Fields and D. M. Knipe, eds.) for the AAV-2 sequence. As used herein, an“AAV ITR” need not have the wild-type nucleotide sequence depicted, butmay be altered, e.g., by the insertion, deletion or substitution ofnucleotides. Additionally, the AAV ITR may be derived from any ofseveral AAV serotypes, including without limitation, AAV-1, AAV-2,AAV-3, AAV-4, AAV-5, AAVX7, etc. Furthermore, 5′ and 3′ ITRs which flanka selected nucleotide sequence in an AAV vector need not necessarily beidentical or derived from the same AAV serotype or isolate, so long asthey function as intended, i.e., to allow for excision and rescue of thesequence of interest from a host cell genome or vector, and to allowintegration of the heterologous sequence into the recipient cell genomewhen AAV Rep gene products are present in the cell.

By “AAV rep coding region” is meant the art-recognized region of the AAVgenome which encodes the replication proteins of the virus which arecollectively required for replicating the viral genome and for insertionof the viral genome into a host genome during latent infection, orfunctional homologues thereof such as the human herpesvirus 6 (HHV-6)rep gene which is also known to mediate AAV-2 DNA replication (Thomsonet al. (1994) Virology 204:304-311). Thus, the rep coding regionincludes at least the genes encoding for AAV Rep 78 and Rep 68 (the“long forms of Rep”), and Rep 52 and Rep 40 (the “short forms of Rep”),or functional homologues thereof. For a further description of the AAVrep coding region, see, e.g., Muzyczka, N. (1992) Current Topics inMicrobiol. and Immunol. 158:97-129; and Kotin, R. M. (1994) Human GeneTherapy 5:793-801. The rep coding region, as used herein, can be derivedfrom any viral serotype, such as the AAV serotypes described above. Theregion need not include all of the wild-type genes but may be altered,e.g., by the insertion, deletion or substitution of nucleotides, so longas the rep genes present provide for sufficient integration functionswhen expressed in a suitable recipient cell.

By “AAV cap coding region” is meant the art-recognized region of the AAVgenome which encodes the coat proteins of the virus which arecollectively required for packaging the viral genome. Thus, the capcoding region includes at least the genes encoding for the coat proteinsVP1, VP2 and VP3. For a further description of the cap coding region,see, e.g., Muzyczka, N. (1992) Current Topics in Microbiol. and Immunol.158:97-129; and Kotin, R. M. (1994) Human Gene Therapy 5:793-801. TheAAV cap coding region, as used herein, can be derived from any AAVserotype, as described above. The region need not include all of thewild-type cap genes but may be altered, e.g., by the insertion, deletionor substitution of nucleotides, so long as the genes provide forsufficient packaging functions when present in a host cell along with anAAV vector.

By an “AAV vector” is meant a vector derived from an adeno-associatedvirus serotype, including without limitation, AAV-1, AAV-2, AAV-3,AAV-4, AAV-5, AAVX7, etc. AAV vectors can have one or more of the AAVwild-type genes deleted in whole or part, preferably the rep and/or capgenes, but retain functional flanking ITR sequences. Functional ITRsequences are necessary for the rescue, replication and packaging of theAAV virion. Thus, an AAV vector is defined herein to include at leastthose sequences required in cis for replication and packaging (e.g.,functional ITRs) of the virus. The ITRs need not be the wild-typenucleotide sequences, and may be altered, e.g., by the insertion,deletion or substitution of nucleotides, so long as the sequencesprovide for functional rescue, replication and packaging.

“AAV helper functions” refer to AAV-derived coding sequences which canbe expressed to provide AAV gene products that, in turn, function intrans for productive AAV replication. Thus, AAV helper functions includeboth of the major AAV open reading frames (ORFs), rep and cap. The Repexpression products have been shown to possess many functions,including, among others: recognition, binding and nicking of the AAVorigin of DNA replication; DNA helicase activity; and modulation oftranscription from AAV (or other heterologous) promoters. The Capexpression products supply necessary packaging functions. AAV helperfunctions are used herein to complement AAV functions in trans that aremissing from AAV vectors.

The term “AAV helper construct” refers generally to a nucleic acidmolecule that includes nucleotide sequences providing AAV functionsdeleted from an AAV vector which is to be used to produce a transducingvector for delivery of a nucleotide sequence of interest. AAV helperconstructs are commonly used to provide transient expression of AAV repand/or cap genes to complement missing AAV functions that are necessaryfor lytic AAV replication; however, helper constructs lack AAV ITRs andcan neither replicate nor package themselves. AAV helper constructs canbe in the form of a plasmid, phage, transposon, cosmid, virus, orvirion. A number of AAV helper constructs have been described, such asthe commonly used plasmids pAAV/Ad and pIM29+45 which encode both Repand Cap expression products. See, e.g., Samulski et al. (1989) J. Virol.63:3822-3828; and McCarty et al. (1991) J. Virol. 65:2936-2945. A numberof other vectors have been described which encode Rep and/or Capexpression products. See, e.g., U.S. Pat. No. 5,139,941.

The term “accessory functions” refers to non-AAV derived viral and/orcellular functions upon which AAV is dependent for its replication.Thus, the term captures proteins and RNAs that are required in AAVreplication, including those moieties involved in activation of AAV genetranscription, stage specific AAV mRNA splicing, AAV DNA-replication,synthesis of Cap expression products and AAV capsid assembly.Viral-based accessory functions can be derived from any of the knownhelper viruses such as adenovirus, herpesvirus (other than herpessimplex virus type-1) and vaccinia virus.

For example, adenovirus-derived accessory functions have been widelystudied, and a number of adenovirus genes involved in accessoryfunctions have been identified and partially characterized. See, e.g.,Carter, B. J. (1990) “Adeno-Associated Virus Helper Functions,” in CRCHandbook of Paarvoviruses, vol. I (P. Tijssen, ed.), and Muzyczka, N.(1992) Curr. Topics. Microbiol. and Immun. 158:97-129. Specifically,early adenoviral gene regions E1a, E2a, E4, VAI RNA and, possibly, E1bare thought to participate in the accessory process. Janik et al. (1981)Proc. Natl. Acad. Sci. USA 78:1925-1929. Herpesvirus-derived accessoryfunctions have been described. See, e.g., Young et al. (1979) Prog. Med.Virol. 25:113. Vaccinia virus-derived accessory functions have also beendescribed. See, e.g., Carter, B. J. (1990), supra., Schlehofer et al.(1986) Virology 152:110-117.

The term “accessory function vector” refers generally to a nucleic acidmolecule that includes nucleotide sequences providing accessoryfunctions. An accessory function vector can be transfected into asuitable host cell, wherein the vector is then capable of supporting AAVvirion production in the host cell. Expressly excluded from the term areinfectious viral particles as they exist in nature, such as adenovirus,herpesvirus or vaccinia virus particles. Thus, accessory functionvectors can be in the form of a plasmid, phage, transposon or cosmid.

By “capable of supporting efficient rAAV virion production” is meant theability of an accessory function vector or system to provide accessoryfunctions that are sufficient to complement rAAV virion production in aparticular host cell at a level substantially equivalent to or greaterthan that which could be obtained upon infection of the host cell withan adenovirus helper virus. Thus, the ability of an accessory functionvector or system to support efficient rAAV virion production can bedetermined by comparing rAAV virion titers obtained using the accessoryvector or system with titers obtained using infection with an infectiousadenovirus. More particularly, an accessory function vector or systemsupports efficient rAAV virion production substantially equivalent to,or greater than, that obtained using an infectious adenovirus when theamount of virions obtained from an equivalent number of host cells isnot more than about 200 fold less than the amount obtained usingadenovirus infection, more preferably not more than about 100 fold less,and most preferably equal to, or greater than, the amount obtained usingadenovirus infection.

By “recombinant virus” is meant a virus that has been geneticallyaltered, e.g., by the addition or insertion of a heterologous nucleicacid construct into the particle.

By “AAV virion” is meant a complete virus particle, such as a wild-type(wt) AAV virus particle (comprising a linear, single-stranded AAVnucleic acid genome associated with an AAV capsid protein coat). In thisregard, single-stranded AAV nucleic acid molecules of eithercomplementary sense, e.g., “sense” or “antisense” strands, can bepackaged into any one AAV virion and both strands are equallyinfectious.

A “recombinant AAV virion,” or “rAAV virion” is defined herein as aninfectious, replication-defective virus composed of an AAV proteinshell, encapsidating a heterologous nucleotide sequence of interestwhich is flanked on both sides by AAV ITRs. A rAAV virion is produced ina suitable host cell which has had an AAV vector, AAV helper functionsand accessory functions introduced therein. In this manner, the hostcell is rendered capable of encoding AAV polypeptides that are requiredfor packaging the AAV vector (containing a recombinant nucleotidesequence of interest) into infectious recombinant virion particles forsubsequent gene delivery.

The term “transfection” is used to refer to the uptake of foreign DNA bya cell, and a cell has been “transfected” when exogenous DNA has beenintroduced inside the cell membrane. A number of transfection techniquesare generally known in the art. See, e.g., Graham et al. (1973)Virology, 52:456, Sambrook et al. (1989) Molecular Cloning, a laboratorymanual, Cold Spring Harbor Laboratories, New York, Davis et al. (1986)Basic Methods in Molecular Biology, Elsevier, and Chu et al. (1981) Gene13:197. Such techniques can be used to introduce one or more exogenousDNA moieties, such as a nucleotide integration vector and other nucleicacid molecules, into suitable host cells.

The term “host cell” denotes, for example, microorganisms, yeast cells,insect cells, and mammalian cells, that can be, or have been, used asrecipients of an AAV helper construct, an AAV vector plasmid, anaccessory function vector, or other transfer DNA. The term includes theprogeny of the original cell which has been transfected. Thus, a “hostcell” as used herein generally refers to a cell which has beentransfected with an exogenous DNA sequence. It is understood that theprogeny of a single parental cell may not necessarily be completelyidentical in morphology or in genomic or total DNA complement as theoriginal parent, due to natural, accidental, or deliberate mutation.

As used herein, the term “cell line” refers to a population of cellscapable of continuous or prolonged growth and division in vitro. Often,cell lines are clonal populations derived from a single progenitor cell.It is further known in the art that spontaneous or induced changes canoccur in karyotype during storage or transfer of such clonalpopulations. Therefore, cells derived from the cell line referred to maynot be precisely identical to the ancestral cells or cultures, and thecell line referred to includes such variants.

The term “heterologous” as it relates to nucleic acid sequences such ascoding sequences and control sequences, denotes sequences that are notnormally joined together, and/or are not normally associated with aparticular cell. Thus, a “heterologous” region of a nucleic acidconstruct or a vector is a segment of nucleic acid within or attached toanother nucleic acid molecule that is not found in association with theother molecule in nature. For example, a heterologous region of anucleic acid construct could include a coding sequence flanked bysequences not found in association with the coding sequence in nature.Another example of a heterologous coding sequence is a construct wherethe coding sequence itself is not found in nature (e.g., syntheticsequences having codons different from the native gene). Similarly, acell transformed with a construct which is not normally present in thecell would be considered heterologous for purposes of this invention.Allelic variation or naturally occurring mutational events do not giverise to heterologous DNA, as used herein.

A “coding sequence” or a sequence which “encodes” a particular protein,is a nucleic acid sequence which is transcribed (in the case of DNA) andtranslated (in the case of mRNA) into a polypeptide in vitro or in vivowhen placed under the control of appropriate regulatory sequences. Theboundaries of the coding sequence are determined by a start codon at the5′ (amino) terminus and a translation stop codon at the 3′ (carboxy)terminus. A coding sequence can include, but is not limited to, cDNAfrom prokaryotic or eukaryotic MRNA, genomic DNA sequences fromprokaryotic or eukaryotic DNA, and even synthetic DNA sequences. Atranscription termination sequence will usually be located 3′ to thecoding sequence.

A “nucleic acid” sequence refers to a DNA or RNA sequence. The termcaptures sequences that include any of the known base analogues of DNAand RNA such as, but not limited to 4-acetylcytosine,8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine,5-(carboxyhydroxylmethyl) uracil, 5-fluorouracil, 5-bromouracil,5-carboxymethylaminomethyl-2-thiouracil,5-carboxymethylaminomethyluracil, dihydrouracil, inosine,N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-.D-mannosylqueosine,5′-methoxycarbonylmethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine,2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,5-methyluracil, N-uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and2,6-diaminopurine.

The term DNA “control sequences” refers collectively to promotersequences, polyadenylation signals, transcription termination sequences,upstream regulatory domains, origins of replication, internal ribosomeentry sites (“IRES”), enhancers, and the like, which collectivelyprovide for the replication, transcription and translation of a codingsequence in a recipient cell. Not all of these control sequences needalways be present so long as the selected coding sequence is capable ofbeing replicated, transcribed and translated in an appropriate hostcell.

The term “promoter region” is used herein in its ordinary sense to referto a nucleotide region comprising a DNA regulatory sequence, wherein theregulatory sequence is derived from a gene which is capable of bindingRNA polymerase and initiating transcription of a downstream(3′-direction) coding sequence.

“Operably linked” refers to an arrangement of elements wherein thecomponents so described are configured so as to perform their usualfunction. Thus, control sequences operably linked to a coding sequenceare capable of effecting the expression of the coding sequence. Thecontrol sequences need not be contiguous with the coding sequence, solong as they function to direct the expression thereof. Thus, forexample, intervening untranslated yet transcribed sequences can bepresent between a promoter sequence and the coding sequence and thepromoter sequence can still be considered “operably linked” to thecoding sequence.

By “isolated” when referring to a nucleotide sequence, is meant that theindicated molecule is present in the substantial absence of otherbiological macromolecules of the same type. Thus, an “isolated nucleicacid molecule which encodes a particular polypeptide” refers to anucleic acid molecule which is substantially free of other nucleic acidmolecules that do not encode the subject polypeptide; however, themolecule may include some additional bases or moieties which do notdeleteriously affect the basic characteristics of the composition.

For the purpose of describing the relative position of nucleotidesequences in a particular nucleic acid molecule throughout the instantapplication, such as when a particular nucleotide sequence is describedas being situated “upstream,” “downstream,” “3′,” or “5′” relative toanother sequence, it is to be understood that it is the position of thesequences in the “sense” or “coding” strand of a DNA molecule that isbeing referred to as is conventional in the art.

“Homology” refers to the percent of identity between two polynucleotideor two polypeptide moieties. The correspondence between the sequencefrom one moiety to another can be determined by techniques known in theart. For example, homology can be determined by a direct comparison ofthe sequence information between two polypeptide molecules by aligningthe sequence information and using readily available computer programs.Alternatively, homology can be determined by hybridization ofpolynucleotides under conditions which allow for the formation of stableduplexes between homologous regions, followed by digestion withsingle-stranded-specific nuclease(s), and size determination of thedigested fragments. Two DNA, or two polypeptide sequences are“substantially homologous” to each other when at least about 80%,preferably at least about 90%, and most preferably at least about 95% ofthe nucleotides or amino acids match over a defined length of themolecules, as determined using the methods above.

A “functional homologue,” or a “functional equivalent” of a givenpolypeptide includes molecules derived from the native polypeptidesequence, as well as recombinantly produced or chemically synthesizedpolypeptides which function in a manner similar to the referencemolecule to achieve a desired result. Thus, a functional homologue ofAAV Rep68 or Rep78 encompasses derivatives and analogues of thosepolypeptides—including any single or multiple amino acid additions,substitutions and/or deletions occurring internally or at the amino orcarboxy termini thereof—so long as integration activity remains.

A “functional homologue,” or a “functional equivalent” of a givenadenoviral nucleotide region includes similar regions derived from aheterologous adenovirus serotype, nucleotide regions derived fromanother virus or from a cellular source, as well as recombinantlyproduced or chemically synthesized polynucleotides which function in amanner similar to the reference nucleotide region to achieve a desiredresult. Thus, a functional homologue of an adenoviral VA RNA gene regionor an adenoviral E2a gene region encompasses derivatives and analoguesof such gene regions—including any single or multiple nucleotide baseadditions, substitutions and/or deletions occurring within the regions,so long as the homologue retains the ability to provide its inherentaccessory function to support AAV virion production at levels detectableabove background.

B. General Methods

Central to the present invention is the development of accessoryfunction systems which allow for the efficient production of rAAVvirions in the absence of infection with a helper virus. Unlike priorproduction methods, accessory functions are provided by introducing oneor more vectors, such as plasmids, which contain genes required forcomplementing rAAV virion production, into a host cell. In this manner,the present accessory function systems can support the production ofcommercially significant levels of rAAV virions without significantlevels of contaminating helper virus particles, or other contaminatingvirus products (e.g., the adenoviral fiber protein). Efficientproduction of rAAV virions is achieved when rAAV virion yields areobtained at levels that are not lower than about 200 fold less thanlevels obtained when using adenovirus type-2 infection to provide theaccessory functions.

The accessory functions are provided on one or more vectors. Thevector(s) will include adenoviral-derived nucleotide sequences necessaryfor rAAV virion production. As explained further below, the sequencespresent on the accessory function construct(s) will be determined by thehost cell used and can include E1a, E1b, E2a, E4 and VA RNA regions.

While not being bound by any particular theory, the accessory functionsprovided by the adenovirus E1b, E2a, and E4 early genes are thought tobe required in AAV DNA replication. The accessory functions provided bythe adenovirus E1b, E4 and VA RNA gene regions appear to participate inpost-transcriptional or translational events in the AAV life cycle. Inregard to the accessory functions provided by E4, only the E4 34 kDprotein encoded by open reading frame 6 (ORF 6) of the E4 coding regionis clearly required for AAV replication. The accessory functionsprovided by the adenovirus gene region E1a are thought to be required asmodulators to activate transcription or expression of the otheradenovirus gene regions, including E1b, E2a, E4 and VA RNA.

The accessory function vectors of the invention can alternativelyinclude one or more polynucleotide homologues which replace theadenoviral gene sequences, so long as each homologue retains the abilityto provide the accessory functions of the replaced adenoviral gene.Thus, homologous nucleotide sequences can be derived from anotheradenoviral serotype (e.g., adenovirus type-2), from another helper virusmoiety (e.g. a herpesvirus or vaccinia virus), or can be derived fromany other suitable source.

Further, accessory function vectors constructed according to theinvention can be in the form of a plasmid, phage, transposon or cosmid.Alternatively, the vector can be in the form of one or more linearizedDNA or RNA fragments which, when associated with the appropriate controlelements and enzymes, can be transcribed or expressed in a host cell toprovide accessory functions. All of the above-described vectors can bereadily introduced into a suitable host cell using transfectiontechniques that are known in the art. Such transfection methods havebeen described, including calcium phosphate co-precipitation (Graham etal. (1973) Virol. 52:456-467), direct micro-injection into culturedcells (Capecchi, M. R. (1980) Cell 22:479-488), electroporation(Shigekawa et al. (1988) BioTechniques 6:742-751), liposome mediatedgene transfer (Mannino et al. (1988) BioTechniques 6:682-690),lipid-mediated transfection (Felgner et al. (1987) Proc. Natl. Acad.Sci. USA 84:7413-7417), and nucleic acid delivery using high-velocitymicroprojectiles (Klein et al. (1987) Nature 327:70-73).

Accessory function vectors can be engineered using conventionalrecombinant techniques. Particularly, nucleic acid molecules can bereadily assembled in any desired order by inserting one or moreaccessory function nucleotide sequences into a construct, such as byligating restriction fragments into a cloning vector using polylinkeroligonucleotides or the-like. The newly formed nucleic acid molecule canthen be excised from the vector and placed in an appropriate expressionconstruct using restriction enzymes or other techniques that are wellknown in the art.

More particularly, selected adenoviral genes or gene regions (e.g., E1a,E1b, E2a, E4 and VA RNA), or functional homologues thereof, can beexcised from a viral genome, or from a vector containing the same, andinserted into a suitable vector either individually, or linked together,to provide an accessory function construct using standard ligationtechniques such as those described in Sambrook et al., supra. Referringto FIG. 2, one such construct can be engineered to include four nucleicacid molecules derived from the adenovirus type-5 genome: a VARNA-containing region; an E2a-containing region; an E4-containing regionand an E1a, E1b-containing region. Specifically, FIG. 2 shows: a 1,724bp SalI-HinDIII VA RNA-containing fragment (corresponding to thenucleotides spanning positions about 9,831 to about 11,555 of theadenovirus type-2 genome); a 5,962 bp SrfI-BamHI E2a-containing fragment(corresponding to the nucleotides spanning positions about 21,606 toabout 27,568 of the adenovirus type-2 genome); a 3,669 bp HphI-HinDIIIE4-containing fragment (corresponding to the nucleotides spanningpositions about 32,172 to about 36,841 of the adenovirus type-2 genome);and a 4,102 bp BsrGI-Eco47III E1a-, E1b-containing fragment(corresponding to the nucleotides spanning positions about 192 to about4294 of the adenovirus type-2 genome), wherein the nucleic acidmolecules are ligated together to provide a complete complement ofaccessory functions in a single accessory function construct. Ligationscan be accomplished in 20 mM Tris-Cl pH 7.5, 10 mM MgCl₂, 10 mM DTT, 33ug/ml BSA, 10 mM-50 mM NaCl, and either 40 uM ATP, 0.01-0.02 (Weiss)units T4 DNA ligase at 0° C. (for “sticky end” ligation) or 1 mM ATP,0.3-0.6 (weiss) units T4 DNA ligase at 14° C. (for “blunt end”ligation). Intermolecular “sticky end” ligations are usually performedat 30-100 μg/ml total DNA concentrations (5-100 nM total endconcentration). The assembled molecule can then be readily inserted intoan expression vector which is capable of transferring the accessoryfunction construct between cells.

In the alternative, nucleic acid molecules comprising one or moreaccessory functions can be synthetically derived, using a combination ofsolid phase direct oligonucleotide synthesis chemistry and enzymaticligation methods which are conventional in the art. Synthetic sequencesmay be constructed having features such as restriction enzyme sites, andcan be prepared in commercially available oligonucleotide synthesisdevices such as those devices available from Applied Biosystems, Inc.(Foster City, Calif.) using the phosphoramidite method. See, e.g.,Beaucage et al. (1981) Tetrahedron Lett. 22:1859-1862. The nucleotidesequence of the adenovirus type-2 genome is generally known, and ispublicly available (e.g., as GeneBank Reference Name: ADRCG, AccessionNumber: J01917; and as NCBI Identification Number: 209811). Thenucleotide sequence of the adenovirus type-5 genome is believed to be99% homologous to the adenovirus type-2 genome. Preferred codons forexpression of the synthetic molecule in mammalian cells can also bereadily synthesized. Complete nucleic acid molecules are then assembledfrom overlapping oligonucleotides prepared by the above methods. See,e.g., Edge, Nature (1981) 292:756; Nambair et al. Science (1984)223:1299; Jay et al. J. Biol. Chem. (1984) 259:6311.

When adenoviral gene regions are used in the vectors of the invention toprovide accessory functions, those regions will be operably linked tocontrol sequences that direct the transcription or expression thereof.Such control sequences can comprise those adenoviral control sequencesnormally associated with the gene regions in the wild-type adenoviralgenome. Alternatively, heterologpus control sequences can be employedwhere desired. Useful heterologous promoter sequences include thosederived from sequences encoding mammalian genes or viral genes. Examplesinclude, but are not limited to, homologous adenoviral promoters, theSV40 early promoter, mouse mammary tumor virus LTR promoter; adenovirusmajor late promoter (Ad MLP); a herpes simplex virus (HSV) promoter, acytomegalovirus (CMV) promoter (e.g., the CMV immediate early promoterregion), a rous sarcoma virus (RSV) promoter, synthetic promoters,hybrid promoters, and the like. In addition, sequences derived fromnonviral genes, such as the murine metallothionein gene, will also finduse herein. Such promoter sequences are commercially available from,e.g., Stratagene (San Diego, Calif.).

Furthermore, the vectors of the present invention can be constructed toalso include selectable markers. Suitable markers include genes whichconfer antibiotic resistance or sensitivity, or impart color, or changethe antigenic characteristics when cells which have been transfectedwith the nucleic acid constructs are grown in an appropriate selectivemedium. Particular selectable marker genes useful in the practice of theinvention include the hygromycin B resistance gene (encodingAminoglycoside phosphotranferase (APH)) that allows selection inmammalian cells by conferring resistance to G418 (available from Sigma,St. Louis, Mo.). Other suitable markers are known to those of skill inthe art.

Accessory function vectors containing a full complement of theadenoviral accessory function genes or gene regions (e.g., E1a, E1b,E2a, E4, VA RNA, and/or functional homologues thereof) can be used tosupply accessory functions to a host cell, including those cells notpermissive for helper viruses (e.g., not infectable by a helper virussuch as an adenovirus or not capable of supporting helper virusreplication). In this manner, rAAV virion production can be carried outin a wide range of host cells, including those which were previouslyrefractive to supporting such production.

In the alternative, accessory function vectors can be constructed tocontain less than a full complement of accessory functions. Such vectorscan be used in a cell that is already capable of supplying one or moreaccessory functions, for example, in a cell that supplies one or moreaccessory functions either inherently (e.g., where the cell expresses anaccessory function homologue) or due to a transformation event.Accessory function vectors containing less than a full complement ofaccessory functions can also be used in combination with other ancillaryaccessory function constructs.

Particularly, suitable host cells can be engineered using ordinaryrecombinant techniques to produce cells that provide one or moreaccessory functions. For example, the human cell line 293 is a humanembryonic kidney cell line that has been transformed with adenovirustype-5 DNA fragments (Graham et al. (1977) J. Gen. Virol. 36:59), andexpresses the adenoviral E1a and E1b genes (Aiello et al. (1979)Virology 94:460). The 293 cell line is readily transfected, and providesa particularly convenient platform in which to produce rAAV virions.Thus, in one particularly preferred embodiment of the invention, anaccessory function vector is provided having only the adenoviral E2a, E4and VA RNA gene regions, or functional homologues thereof.

Referring to FIG. 1, one such construct can be engineered to includethree nucleic acid molecules derived from the adenovirus type-5 genome:a 1,724 bp SalI-HinDIII VA RNA-containing fragment (corresponding to thenucleotides spanning positions about 9,831 to about 11,555 of theadenovirus-type-2 genome); a 5,962 bp SrfI-BamHI E2a-containing fragment(corresponding to the nucleotides spanning positions about 21,606 toabout 27,568 of the adenovirus type-2 genome); and a 3,669 bpHphI-HinDIII E4-containing fragment (corresponding to the nucleotidesspanning positions about 32,172 to about 36,841 of the adenovirus type-2genome), wherein the nucleic acid molecules are ligated together toprovide a truncated complement of accessory functions in a singleaccessory function construct.

Referring to FIG. 3, an alternative construct can be engineered toinclude three nucleic acid molecules derived from the adenovirus type-2genome: a 732 bp EcoRV-SacII VA RNA-containing fragment (correspondingto the nucleotides spanning-positions about 10,423-11,155 of theadenovirus type-2 genome); a 5,962 bp SrfI-KpnI E2a-containing fragment(corresponding to the nucleotides spanning positions about 21,606 toabout 27,568 of the adenovirus type-2 genome); and a 3,192 bp modifiedSrfI-SpeI E4 ORF6-containing fragment (corresponding to the nucleotidesspanning positions about 32,644 to about 34,120 of the adenovirus type-2genome. The nucleic acid molecules are ligated together to provide aneven further truncated complement of accessory functions in a singleaccessory function construct.

These vectors can be constructed as described above using recombinantand/or synthetic techniques, and can include a variety of ancillarycomponents such as heterologous promoter regions, selectable markers andthe like. Upon transfection into a host 293 cell, the vectors provideaccessory functions that are capable of supporting efficient rAAV virionproduction.

Once engineered, the accessory function vectors of the present inventioncan be used in a variety of systems for rAAV virion production. Forexample, suitable host cells that have been transfected with one or moreaccessory function vectors are thereby rendered capable of producingrAAV virions when co-transfected with an AAV vector and an AAV helperconstruct capable of being expressed in the cell to provide AAV helperfunctions.

The AAV vector, AAV helper construct and the accessory functionvector(s) can be introduced into the host cell, either simultaneously orserially, using transfection techniques described above.

AAV vectors used to produce rAAV virions for delivery of a nucleotidesequence of interest can be constructed to include one or moreheterologous nucleotide sequences flanked on both ends (5′ and 3′) withfunctional AAV ITRs. In the practice of the invention, an AAV vectorgenerally includes at least one AAV ITR and an appropriate promotersequence suitably positioned relative to a heterologous nucleotidesequence, and at least one AAV ITR positioned downstream of theheterologous sequence. The 5′ and 3′ ITRs need not necessarily beidentical to, or derived from, the same AAV isolate, so long as theyfunction as intended.

Suitable heterologous nucleotide sequences for use in AAV vectorsinclude any functionally relevant nucleotide sequence. Thus, AAV vectorsfor use in the practice of the invention can include any desired genethat encodes a protein that is defective or missing from a recipientcell genome or that encodes a non-native protein having a desiredbiological or therapeutic effect (e.g., an antiviral function), or thesequence can correspond to a molecule having an antisense or ribozymefunction. Suitable genes include, but are not limited to, those used forthe treatment of inflammatory diseases, autoimmune, chronic andinfectious diseases, including such disorders as AIDS, cancer,neurological diseases, cardiovascular disease, hypercholestemia; variousblood disorders including various anemias, thalasemias and hemophilia;genetic defects such as cystic fibrosis, Gaucher's Disease, adenosinedeaminase (ADA) deficiency, emphysema, or the like. A number ofantisense oligonucleotides (e.g., short oligonucleotides complementaryto sequences around the translational initiation site (AUG codon) of anmRNA) that are useful in antisense therapies for cancer, cardiovascular,and viral diseases have been described in the art. See, e.g., Han et al.(1991) Proc. Natl. Acad. Sci. USA 88:4313-4317; Uhlmann et al. (1990)Chem. Rev. 90:543-584; Helene et al. (1990) Biochim. Biophys. Acta.1049:99-125; Agarwal et al. (1988) Proc. Natl. Acad. Sci. USA85:7079-7083; and Heikkila et al. (1987) Nature 328:445-449. For adiscussion of suitable ribozymes, see, e.g., Cech et al. (1992) J. Biol.Chem. 267:17479-17482 and U.S. Pat. No. 5,225,347 to Goldberg et al.

AAV vectors can also include control sequences, such as promoter andpolyadenylation sites, as well as selectable markers or reporter genes,enhancer sequences, and other control elements which allow for theinduction of transcription. Such AAV vectors can be constructed usingtechniques well known in the art. See, e.g., U.S. Pat. No. 5,173,414;International Publication Numbers WO 92/01070 (published 23 Jan. 1992)and WO 93/03769 (published 4 Mar. 1993); Lebkowski et al. (1988) Molec.Cell. Biol. 8:3988-3996; Vincent et al. (1990) Vaccines 90 (Cold SpringHarbor Laboratory Press); Carter, B. J. (1992) Current Opinion inBiotechnology 3:533-539; Muzyczka, N. (1992) Current Topics inMicrobiol. and Immunol. 158:97-129; Kotin, R. M. (1994) Human GeneTherapy 5:793-801; Shelling and Smith (1994) Gene Therapy 1:165-169; andZhou et al. (1994) J. Exp. Med. 179:1867-1875.

In the methods of the-invention, AAV helper constructs are used tocomplement AAV functions deleted from an AAV vector. A number ofsuitable AAV helper constructs have been described, including, e.g., theplasmids pAAV/Ad and pIM29+45 which encode both rep and cap expressionproducts (see, e.g., Samulski et al. (1989) J. Virol. 63:3822-3828 andMcCarty et al. (1991) J. Virol. 65:2936-2945). Complementing AAV helperfunctions in this manner to support rAAV virion production is anart-accepted technique. However, due to homologous recombination eventsbetween the AAV ITR sequences present in the AAV vector and the AAVhelper function sequences present in the helper construct, suchtechniques also generate contaminating wild-type AAV virions in the rAAVvirion stocks. The presence of wild-type AAV particles in AAV-basedvector systems could potentially lead to unintentional spread ofrecombinant AAV virions, and may interfere with the efficient expressionof foreign genes.

C. Experimental

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for.

Plasmid Construction:

Plasmid JM17 (McGrory et al. (1988) Virology 163:614-617), whichcomprises a circularized isolate of adenovirus type-5 with the plasmidvector pBR322X inserted at the unique XbaI site at the end of theadenovirus E1a gene, was used as a source of adenovirus genes. Neitheradenovirus type-5 nor pJM17 has been completely sequenced. Accordingly,the sizes of the adenovirus type-5 fragments described below have beenapproximated. Since adenovirus type-2 has been fully sequenced, andadenovirus types 2 and 5 are thought to be approximately 99% homologous,the adenovirus type-5 fragment sizes are approximated based uponcorresponding adenovirus type-2 fragments.

Plasmid pBSII-VA RNAs (ATCC Accession Number 98233) was constructed asfollows. The approximately 5,324 bp HinDIII-HinDIII fragment (containingthe adenovirus type-5 VA RNA I and II-coding regions) was obtained fromthe plasmid pJM17. The 5,324 bp fragment was inserted into the plasmidvector pBSII s/k− (obtained from Stratagene) at the HinDIII site. The5,324 bp fragment corresponds to the nucleotides extending from position6,231 to 11,555 (HinDIII sites) of the adenovirus type-2 genome(publicly available, e.g., as GeneBank Reference Name: ADRCG, AccessionNumber: J01917; and NCBI Identification Number: 209811).

Plasmid pBSII-E2a+E4 was constructed as follows. The approximately15,667 bp BamHI-XbaI fragment (containing the adenovirus type-5 E2a, E3and E4 coding regions, adenoviral terminal repeats ligated head to head,and a portion of the adenovirus type-5 E1a coding region) was obtainedfrom the plasmid pJM17. The plasmid vector pBSII s/k− was cut with BamHIand XbaI, and the 15,667 bp fragment was cloned into the subject vector.The 15,667 bp fragment corresponds to the nucleotides extending fromposition 21,606 (a BamHI site) to 35,937 (the distal end of the 3′terminus), and the nucleotides extending from position 1 (the beginningof the 5′ terminus) to 1,336 (an XbaI site) of the adenovirus type-2genome.

Plasmid pBSII-E2a was constructed as follows. The approximately 5,935 bpBamHI-EcoRI fragment (containing the adenovirus type-5 E2a codingregion) was obtained from the plasmid pJM17. The plasmid vector PBSIIs/k− was cut with BamHI and EcoRI, and the 5,935 bp fragment was clonedinto the subject vector. The 5,935 bp fragment corresponds to thenucleotides extending from position 15,403 (a BamHI site) to 21,338 (anEcoRI site) of the adenovirus type-2 genome.

Plasmid pBSII-E4 was constructed as follows. The approximately 5,111 bpXhoI-XbaI fragment (containing the adenovirus type-5 E4 coding region,adenoviral terminal repeats ligated head to head, and a portion of theadenovirus type-5 E1 coding region) was obtained from pJM17. The plasmidvector pBSII s/k− was cut with XhoI and XbaI, and the 5,111 bp fragmentwas cloned into the subject vector. The 5,111 bp fragment corresponds tothe nucleotides extending from position 29,788 (an XhoI site) to 35,937(the end of the 3′ terminus), and the nucleotides extending fromposition 1 (the beginning of the 5′ terminus) to 1,336 (an XbaI site) ofthe adenovirus type-2 genome.

Plasmid pWadhlacZ was constructed as follows. The plasmid pUC119(GeneBank Reference Name: U07649, GeneBank Accession Number: U07649) waspartially digested with AflIII and BspHI, blunt-end modified with theklenow enzyme, and then ligated to form a circular 1732 bp plasmidcontaining the bacterial origin and the amp gene only (the polylinkerand F1 origin was removed). The blunted and ligated AflIII and BspHIjunction forms a unique NspI site. The 1732 bp plasmid was cut withNspI, blunt-end modified with T4 polymerase, and a 20 bp HinDIII-HinCIIfragment (blunt-end modified with the klenow enzyme) obtained from thepUC119 polylinker was ligated into the blunted NspI site of the plasmid.The HinDIII site from the blunted polylinker was regenerated, and thenpositioned adjacent to the bacterial origin of replication. Theresulting plasmid was then cut at the unique PstI/Sse8387I site, and anSse8387I-PvuII-Sse8387I oligonucleotide (5′-GGCAGCTGCCTGCA-3′ (SEQ IDNO:1)) was ligated in. The remaining unique BspHI site was cut,blunt-end modified with klenow enzyme, and an oligonucleotide containingan AscI linker (5′-GAAGGCGCGCCTTC-3′ (SEQ ID NO:2)) was ligated therein,eliminating the BspHI site. The resulting plasmid was called pWee.

In order to create the pWadhlacZ construct, a CMVlacZ expressioncassette (comprising a nucleotide sequence flanked 5′ and 3′ by AAVITRS, wherein the nucleotide sequence contains the following elements: aCMV promoter, the hGH 1st intron, an adhlacz fragment and an SV40 earlypolyadenylation site) was inserted into the unique PvuII site of pWeeusing multiple steps such that the CMV promoter was arranged proximal tothe bacterial amp gene of pWee.

More particularly, a CMVlacZ expression cassette was derived from theplasmid psub201CMV, which was constructed as follows. An oligonucleotideencoding the restriction enzyme sites:NotI-MluI-SnaBI-AgeI-BstBI-BssHII-NcoI-HpaI-BspEI-PmlI-RsrII-NotI andhaving the following nucleotide sequence:

-   5′-GCGGCCGCACGCGTACGTACCGGTTCGAAGCGCGCACGGCCGACCATGGT    TAACTCCGGACACGTGCGGACCGCGGCCGC-3′ (SEQ ID NO:3) was synthesized and    cloned into the blunt-end modified KasI-EarI site (partial) of    pUC119 to provide a 2757 bp vector fragment. A 653 bp SpeI-SacII    fragment containing a nucleotide sequence encoding a CMV immediate    early promoter was cloned into the SnaBI site of the 2757 bp vector    fragment. Further, a 269 bp PCR-produced BstBI-BstBI fragment    containing a nucleotide sequence encoding the hGH 1st intron which    was derived using the following primers:-   5′-AAAATTCGAACCTGGGGAGAAACCAGAG-3′ (SEQ ID NO:4) and    3′-aaaattcgaacaggtaagcgcccctTTG-5′ (SEQ ID NO:5), was cloned into    the BstBI site of the 2757 bp vector fragment, and a 135 bp    HpaI-BamHI (blunt-end modified) fragment containing the SV40 early    polyadenylation site from the pCMV-β plasmid (obtained from    Clonetech) was cloned into the HpaI site of the subject vector    fragment to result in a plasmid called p1.1c. The p1.1c plasmid was    then cut with NotI to provide a first CMV expression cassette.

The plasmid psub201 (Samulski et al. (1987) J. Virol 61:3096-3101) wascut with XbaI, blunt-end modified, and NotI linkers (5′-TTGCGGCCGCAA-3′(SEQ ID NO:6) were ligated to the ends to provide a vector fragmentcontaining the bacterial origin of replication and an amp gene, whereinthe vector fragment is flanked on both sides by NotI sites. After beingcut with NotI, the first CMV expression cassette was cloned into thepsub201 vector fragment to create psub201CMV. The ITR-bounded expressioncassette from this plasmid was isolated by cutting with PvuII, andligated to pWee after that plasmid was cut with PvuII to create pWCMV.pWCMV was then cut with BssHII (partial), and a 3246 bp fragmentcontaining the adhlacZ gene (a SmaI-DraI nucleotide fragment obtainedfrom the plasmid pCMV-β, having AscI linkers (5′-GAAGGCGCGCCTTC-3′ (SEQID NO:7)) ligated to the ends to provide a 3246 bp fragment) was ligatedinto the BssHII site of pWCMV to obtain the pWadhlacZ construct.

Plasmid pW1909adhlacZ was constructed as follows. A 4723 bp SpeI-EcoRVfragment containing the AAV rep and cap encoding region was obtainedfrom the plasmid pGN1909 (ATCC Accession Number 69871). The pGN1909plasmid is a high efficiency AAV helper plasmid having AAV rep and capgenes with an AAV p5 promoter region that is arranged in the constructto be downstream from its normal position (in the wild type AAV genome)relative to the rep coding region. The 4723 bp fragment was blunt-endmodified, and AscI linkers (5′-GAAGGCGCGCCTTC-3′ (SEQ ID NO:8)) wereligated to the blunted ends. The resultant fragment was then ligatedinto the unique AscI site of pWadhlacZ and oriented such that the AAVcoding sequences were arranged proximal to the bacterial origin ofreplication in the construct.

Plasmid pW620adhlacZ was constructed as follows. A 4439 bp MscI fragmentcontaining the AAV rep and cap encoding region was obtained from theplasmid pSM620 (Samulski et al. (1982) Proc. Natl. Acad. Sci. USA79:2077-2081. The 4439 bp fragment was blunt-end modified, and AscIlinkers (5′-GAAGGCGCGCCTTC-3′ (SEQ ID NO:9)) were ligated to the bluntedends. The resultant fragment was then ligated into the unique AscI siteof pWadhlacZ and oriented such that the AAV coding sequences werearranged proximal to the bacterial origin of replication in theconstruct.

Plasmid pW1909 was constructed as follows. The pW1909adhLacZ plasmid wascut with Sse8387, and the 6506 bp Sse8387I-Sse8387I fragment (containingthe ampicillin resistance gene, the coli 1 origin of replication, andthe AAV helper sequence) was recircularized by intramolecular ligationto provide the pW1909 construct.

The pVlacZ vector plasmid is a modified version of the Sse8387I-Sse8371Ifragment obtained from pW1909adhlacZ that has been cloned into thepUC119 plasmid. The Sse8387I-Sse8371I of pVlacZ differs from theSse8387I-Sse8371I fragment of pW1909 in that all AAV sequences notderived from the AAV inverted terminal repeats (ITRs) have beeneliminated. Plasmid pVlacZ was constructed as follows. Synthetic piecesof DNA, formed by combining AAV serotype 2 base pairs 122-145 with adownstream NotI compatible end, were constructed to provide MscI-NotIfragments containing AAV ITR sequences including all of the D loop. Thesynthetic DNAs were ligated onto both ends of the 4384 bp NotI fragmentfrom the pW1909adhlacZ plasmid. The 4384 bp fragment contains theCMVlacZ sequences. The resulting fragment was then ligated into the 6732bp MscI fragment of pW1909adhlacZ to provide an assembly construct. Theassembly construct was cut with Sse8387I to obtain a 4666 bpSse8387I-Sse8371I fragment (containing the CMVlacZ sequences) and the4666 bp fragment was ligated into pUC119 at the Sse8387I site to obtainthe pVlacZ vector plasmid.

EXAMPLE 1 rAAV Virion Production Using Transfected Adenovirus Genes toSupply Accessory Functions

In order to determine whether adenoviral genes, introduced into asuitable host cell by transfection, are capable of providing accessoryfunctions similar to those provided by an adenoviral infection in thecontext of AAV replication, the following experiment was conducted.

Cells from the stable human cell line, 293 (readily available through,e.g., the ATCC under Accession Number CRL1573), were plated in eight10-cm tissue culture dishes at 1×10⁶ cells/dish to provide 4 duplicateexperimental groups. The cells were then grown at 37° C. to reach 90%confluency over a period of from about 24 to 48 hours prior totransfection. In each group, the plasmid pW1909adhlacZ was used as asource of rescuable AAVlacZ vector and AAV rep and cap coding regions.

Transfections were carried out using a modified calcium phosphate methodwith a total of 20 μg of DNA for a period of 5 hours. More particularly,at 1 to 4 hours prior to transfection, the medium in the tissue cultureplates was replaced with fresh DME/F12 culture medium containing 10%FCS, 1% pen/strep and 1% glutamine. A total of 20 μg of DNA, comprisingone or more vectors, was added to 1 mL of sterile 300 mM CaCl₂, whichwas then added to 1 mL of sterile 2× HBS solution (formed by mixing 280mM NaCl, 50 mM HEPES buffer, 1.5 mM Na₂HPO₄ and adjusting the pH to 7.1with 10 M NaOH) and immediately mixed by gentle inversion. The resultantmixture was pipetted immediately into the 10 cm plates of 90% confluentcells (in 10 mL of the above-described culture medium) and swirled toproduce a homogeneous solution. The plates were transferred to a 5% CO₂incubator and cultured at 37° C. for approximately 5 hours withoutdisturbing. After transfection, the medium was removed from the plates,and the cells washed once with sterile Phosphate buffered saline (PBS).

Adenovirus working stock was prepared by diluting a master stock ofadenovirus type-2 to a concentration of 10⁶ pfu/mL in DME/F12 plus 10%FCS, 1% pen/strep, 1% glutamine and 25 mM sterile HEPES buffer (pH 7.4).

Cell cultures from the first group were transfected with 10 μg each ofthe plasmids pW1909adhlacZ and pBSII s/k−. After the transfectionperiod, the medium was replaced, 10 mL of medium containing adenovirustype-2 at a multiplicity of infection (moi) of 1 was added, and thecultures were incubated at 37° C. for approximately 72 hours.

Cell cultures from the second group were transfected with 10 μg each ofthe plasmids pW1909adhlacZ and pJM17. After the transfection period, themedium was replaced, and the cultures were incubated at 37° C. forapproximately 72 hours.

Cell cultures from the third group were transfected with 10 μg each ofpBSII s/k− and pJM17. After the transfection period, the medium wasreplaced, and the cultures were incubated at 37° C. for approximately 72hours.

Cell cultures from the fourth group were transfected with 10 μg each ofpW1909adhlacZ and pBSII s/k−. After the transfection period, the mediumwas replaced, and the cultures were incubated at 37° C. forapproximately 72 hours.

The cells from each experimental group were then collected, media wasremoved by centrifugation (1000×g for 10 min.), and a 1 mL lysate wasproduced using 3 freeze/thaw cycles (alternating between dry ice-ethanoland 37° C. baths). The lysates were made free of debris bycentrifugation (10,000×g for 10 min). rAAV lacZ virion production wasassessed by titering the freeze/thaw extracts on 293 cells, and assayingfor lacz.

Specifically, 293 cells were plated in 12 well plates (at 1×10⁵ cellsper well) and inoculated with a range of volumes (10-0.01 μL) of theabove-described freeze/thaw lysates and incubated for 24 hours at 37° C.The cells were then fixed and stained by removal of the medium,incubation of the cells for 5 minutes in PBS containing 2% formaldehydeand 0.2% glutaraldehyde, washing once with PBS, and then incubating thecells over-night in PBS containing 5 mM potassium ferrocyanide, 5 mMpotassium ferricyanide, 2 mM magnesium chloride, and 1 mg/ml X-Gal(Sanes et al. (1986) EMBO 5:3133-3142). The rAAV virion titer was thencalculated by quantifying the number of blue cells using lightmicroscopy.

Contaminating infectious adenovirus production was assayed as follows.Samples from the cell lysates were added to 50% confluent 293 cells(cultured in 12 well dishes at 1×10⁵ cells/well), and the cultures werepassaged for 30 days (e.g., the cultures were split 1 to 5, every 3days) or until the culture exhibited 100% CPE due to adenovirusinfection. Cultures were examined daily for CPE, and the day upon whicheach experimental culture showed 100% CPE was noted. Reference 293 cellcultures infected with a range of known amounts of adenovirus type-2(from 0 to 1×10⁷ pfu/culture) were also prepared and treated in the samemanner. A standard curve was then prepared from the data obtained fromthe reference cultures, where the adenovirus pfu number was plottedagainst the day of 100% CPE. The titer of infectious adenovirus type-2in each experimental culture was then readily obtained as determinedfrom the standard curve. The limit of detection in the assay was 100pfu/mL.

The results of the experiment are depicted below in Table 1.

TABLE 1 Transfected ad-2 rAAV Adenovirus Group Plasmids Infection Titer†Titer 1 pW1909lacZ/pBS yes  1 × 10¹⁰ ≧10⁹ pfu/mL 1 pW1909lacZ/pBS yes  1× 10¹⁰ ≧10⁹ pfu/mL 2 pW1909lacZ/pJM17 no 1 × 10⁹ none detected 2pW1909lacZ/pJM17 no 2 × 10⁹ none detected 3 pBS/pJM17 no 0 10⁴ pfu/mL 3pBS/pJM17 no 0 10⁴ pfu/mL 4 pW1909lacZ/pBS no 0 not tested 4pW1909lacZ/pBS no 0 not tested †As determined by the lacZ assay.

As can be seen by the results in Table 1, adenoviral genes introducedinto a host cell by transfection and expressed in the absence ofadenoviral infection can provide accessory functions at a level that isapproximately 20% (e.g., 5 fold less) as effective as the level ofaccessory functions provided by an adenovirus infection.

EXAMPLE 2 Identification of Adenoviral Gene Regions Responsible forAccessory Functions

In order to determine which adenoviral genes or gene regions arenecessary and sufficient in the provision of accessory functions, thefollowing experiment was conducted.

293 cells were plated in twelve 10-cm tissue culture dishes at 1×10⁶cells/dish to provide 6 duplicate experimental groups. The cells werethen grown at 37° C. to reach approximately 90% confluency over a periodof from about 24 to 48 hours prior to transfections. In each group, theplasmid pW1909adhlacZ was used as a source of rescuable AAVlacZ vectorand AAV rep and cap coding regions. Transfections were carried out asdescribed above in Example 1. Adenovirus working stock was also preparedas previously described.

Each of the 293 cell cultures were transfected with the plasmidpW1909adhlacZ and either the plasmid pBSII s/k− (as a control), orvarious combinations of isolated adenoviral genes.

More particularly, cells in the first experimental group wereco-transfected with 5 μg of the plasmid pW1909adhlacZ (to provide AAVhelp functions) and 15 μg of the plasmid pBSII s/k−. After thetransfection period, the medium was replaced, and the cells wereinfected using 10 mL medium containing adenovirus type-2 (moi=1). Thecultures were then incubated at 37° C. for approximately 72 hours.

Cells in the second experimental group were co-transfected with 5 μg ofthe plasmid pW1909adhlacZ (to provide AAV help functions), and 15 μg ofthe plasmid pJM17. After the transfection, the medium was replaced andthe cultures were incubated at 37° C. for approximately 72 hours.

Cells in the third experimental group were co-transfected with 5 μg ofthe plasmid pW1909adhlacZ (to provide AAV help functions), 10 μg of theplasmid pBSII-E2a+E4, and 5 μg of the plasmid pBSII s/k−. After thetransfection, the medium was replaced, and the cultures were incubatedat 37° C. for approximately 72 hours.

Cells in the fourth experimental group were co-transfected with 5 μg ofthe plasmid pW1909adhlacZ (to provide AAV help functions), 10 μg of theplasmid pBSII-E2a+E4, and 5 μg of the plasmid PBSII-VA RNAs. After thetransfection, the medium was replaced, and the cultures were incubatedat 37° C. for approximately 72 hours.

Cells in the fifth experimental group were co-transfected with 5 μg ofthe plasmid pW1909adhlacZ (to provide AAV help functions), 5 μg of theplasmid pBSII-E2a, 5 μg of the plasmid pBSII-E4, and 5 μg of the plasmidpBSII s/k−. After the transfection, the medium was replaced, and thecultures were incubated at 37° C. for approximately 72 hours.

Cells in the sixth experimental group were co-transfected with 5 μg ofthe plasmid pW1909adhlacZ (to provide AAV help functions), 5 μg of theplasmid pBSII-E2a, 5 μg of the plasmid pBSII-E4, and 5 μg of the plasmidPBSII-VA RNAs. After the transfection, the medium was replaced, and thecultures were incubated at 37° C. for approximately 72 hours.

The cells from each experimental group were then collected, media wasremoved by centrifugation (1000×g for 10 min.), and a 1 mL lysate wasproduced using 3 freeze/thaw cycles (alternating between dry ice-ethanoland 37° C. baths). The lysates were made free of debris bycentrifugation (10,000×g for 10 min). rAAV lacZ virion production wasthen assessed using the techniques described in Example 1, and theamount of rAAV genomes was quantified by the following assay method.

50λ of the lysate from each experimental group was added to a 100λaliquot of DMEM medium (available from Sigma, St. Louis, Mo.) containing50 U/mL DNAse I to form assay samples. The samples were incubated at 37°C. for approximately 1 hour, after which 100λ of proteinase K (1 mg/mL)in a 2× proteinase K buffer (20 mM Tris Cl, 20 mM EDTA, 1% SDS, pHadjusted to 8.0) was added to each sample which were then incubated at37° C. for another hour. DNA from the samples was phenol/chloroformextracted, precipitated in EtOH and then collected by centrifugation at5° C. for 15 minutes. The DNA pellets were then redissolved in 200λ TEto provide DNA samples. Dot blot assays were then conducted as follows.Zeta probe® membrane (Bio Rad, Richmond, Calif.) was cut to size andassembled into a dot blot apparatus. The DNA samples were denaturedusing 200λ of a 2× alkaline solution (0.8 M NaOH, 20 mM EDTA), and,after 5 minutes, the membranes were rinsed in a 2×SSC solution for 1minute, dried on filter paper, then baked under vacuum at 80° C. forapproximately 30 minutes. Hybridizations were carried out at 65° C. for30 minutes in hybridization buffer (1 mM EDTA, 40 mM Na₂HPO₄ (pH 7.2),7% SDS). The filters were then washed and autoradiographed forapproximately 20 hours, radioactivity was determined using scintillationcounting.

The results from the experiment are depicted below in Table 2.

TABLE 2 ad-2 rAAV Group Transfected Plasmids Infection Titer† Genomes/mL1 pW1909lacZ yes 2 × 10⁹ 3 × 10¹¹ 2 pW1909lacZ, pJM17 no 2 × 10⁹ 4 ×10¹¹ 3 pW1909lacZ, no 4 × 10⁸ 1 × 10¹¹ pBSII-E2a + E4 4 pW1909lacZ, no 3× 10⁹ 5 × 10¹¹ pBSII-E2a + E4, pBSII-VA RNAs 5 pW1909lacZ, no 2 × 10⁸ 1× 10¹¹ pBSII-E2a, pBSII-E4 6 pW1909lacZ, no 2 × 10⁹ 4 × 10¹¹ pBSII-E2a,pBSII-E4, pBSII-VA RNAs †As determined by the lacZ assay.

As can be seen by the results depicted in Table 2, isolated adenoviralgene regions can be successfully transfected into host cells to provideaccessory functions that are necessary and sufficient for rAAV virionreplication. Further, the results obtained with groups 3 and 5 indicatethat the adenoviral VA RNA region is not essential for the replicationof rAAV virions; however, the region is needed to obtain rAAV titerscomparable to those obtained using adenoviral infection.

EXAMPLE 3 Correlation of Adenoviral VA RNA Dosage to rAAV VirionProduction

In order to investigate whether a correlation exists between the amountof transfected adenoviral VA RNA gene region supplied to a host cell,and the level of accessory functions provided to complement rAAVreplication in the host cell, the following experiment was carried out.

293 cells were plated in 10-cm tissue culture dishes at 1×10⁶cells/dish, and were cultured at 37° C. to reach approximately 90%confluency over a period of from about 24 to 48 hours prior totransfections. Transfections were carried out as described above inExample 1.

Specifically, 293 cells were transfected with 5 μg of the plasmidpW1909adhlacZ, 10 μg of the plasmid pBSII-E2a+E4 and from 0 to 25 μg ofthe plasmid pBSII-VA RNAs to vary the molar ratio of the VA RNA bearingplasmid (relative to the other plasmids) over the range of 0 to 5. Afterthe transfection period, the medium was exchanged, and the cells wereincubated at 37° C. for approximately 72 hours.

Cells from each experimental group were then collected, media wasremoved by centrifugation (1000×g for 10 min.), and a 1 mL lysate wasproduced using 3 freeze/thaw cycles (alternating between dry ice-ethanoland 37° C. baths). The lysates were made free of debris bycentrifugation (10,000×g for 10 min). AAV lacZ vector production wasthen assessed using the techniques described in Example 1. The resultsfrom the experiment are depicted below in Table 3.

TABLE 3 Amount of pBSII-VA RNAs Molar Ratio of pBSII-VA Transfected (μg)RNAs/other plasmids rAAV Titer† 0 0 3 × 10⁸ 1 0.2 6 × 10⁹ 5 1  1 × 10¹⁰10 2 7 × 10⁹ 25 5 5 × 10⁹ †As determined by the lacZ assay.

As can be seen from Table 3, although adenoviral VA RNAs are needed toobtain rAAV virion production at levels substantially equivalent tothose obtained with adenoviral infection, variations in the ratio of VARNA/other adenoviral gene regions over the range investigated does notsignificantly effect rAAV virion production.

EXAMPLE 4 Demonstration of the Requirement for Adenoviral E2a, E4 and VARNA Gene Regions in Accessory Functions

In order to establish the relative contributions of the adenoviral E2a,E4 and VA RNA gene regions in the provision of accessory functions forrAAV virion production, the following experiment was carried out.

293 cells were plated in 10-cm tissue culture dishes at 1×10⁶cells/dish, and were cultured at 37° C. to reach approximately 90%confluency over a period of from about 24 to 48 hours prior toco-transfections. All transfections were carried out as described abovein Example 1.

The 293 cell cultures were co-transfected with 5 g of the plasmidpW1909adhlacZ, and 15 μg (total) of either all, or paired combinationsof the following plasmids: pBSII-E2a; pBSII-E4; and pBSII-VA RNAs toprovide cultures in which each of the adenoviral gene regions encodingE2a, E4 and VA RNA were eliminated from a co-transfection. After thetransfection period, the media was replaced, and the cells wereincubated at 37° C. for approximately 72 hours.

The cells from each co-transfection group were then collected, media wasremoved by centrifugation (1000×g for 10 min.), and a 1 mL lysate wasproduced-using 3 freeze/thaw cycles (alternating between dry ice-ethanoland 37° C. baths). The lysates were made free of debris bycentrifugation (10,000×g for 10 min). rAAV lacz virion production wasthen assessed using the techniques described in Example 1. The resultsfrom the experiment are depicted below in Table 4.

TABLE 4 Co-transfection with Helper Plasmids Encoding E2a E4 VA RNAsrAAV Titer† + + + 5 × 10⁹ − + + 5 × 10⁵ + − + 6 × 10⁷ + + − 7 × 10⁸ − −− 0 †As determined by the lacZ assay.

As can be seen by the results depicted in Table 4, each of theadenoviral gene regions E2a, E4 and VA RNA are not absolutely essentialto provide the accessory functions needed to support rAAV virionproduction; however, all of the gene regions are needed to producelevels of rAAV virions comparable to those obtained with adenoviralinfection. More particularly, omission of the VA RNA containing plasmidfrom the co-transfection resulted in a 7 fold drop in rAAV virionproduction (7×10⁸ functional units per 10 cm dish). rAAV virionproduction was even more severely affected by omission of the E4- andE2a-containing plasmids from the co-transfection. Omission of the E4construct resulted in an 83 fold drop in production (6×10⁷ functionalunits per 10 cm dish), and omission of the E2 construct resulted in a10,000 fold drop in rAAV virion production (5×10⁵ functional units per10 cm dish).

EXAMPLE 5 Comparison of rAAV Virion Production Using PW1909adhlacZ orpW620adhlacZ Based AAV Help

In order to compare the efficiency of rAAV virion production in a hostcell using non-viral accessory function systems with AAV helperconstructs containing either wild-type or modified AAV help functions(AAV rep and cap coding regions), the following experiment was carriedout.

293 cells were plated in 10-cm tissue culture dishes at 1×10⁶cells/dish, and were cultured at 37° C. to reach approximately 90%confluency over a period of from about 24 to 48 hours prior toco-transfections. All transfections were carried out as described abovein Example 1.

A first set of 293 cell cultures was co-transfected with 5 μg of theplasmid pW620adhlacZ (containing wild-type AAV help functions), 10 μg ofthe plasmid pBSII-E2a+E4 and 5 μg of the plasmid pBSII-VA RNAs. A secondset of 293 cultures was co-transfected with 5 μg of the plasmidpW1909adhlacZ (containing modified AAV help functions), 10 μg of theplasmid PBSII-E2a+E4 and 5 μg of the plasmid PBSII-VA RNAs. After thetransfections, the media was replaced, and the cultures were incubatedat 37° C. for approximately 72 hours.

Cells from each co-transfection group were then collected, media wasremoved by centrifugation (1000×g for 10 min.), and a 1 mL lysate wasproduced using 3 freeze/thaw cycles (alternating between dry ice-ethanoland 37° C. baths). The lysates were made free of debris bycentrifugation (10,000×g for 10 min). rAAV lacZ virion production wasthen assessed using the techniques described in Example 1. The resultsfrom the experiment are depicted below in Table 5.

TABLE 5 Vector/Helper Construct rAAV Titer† pW1909adhlacZ 5 × 10⁹pW620adhlacZ 4 × 10⁹ †As determined by the lacZ assay.

As can be seen from the results depicted in Table 5, both the wild-typeand modified forms of AAV help supported rAAV virion production atapproximately the same level.

EXAMPLE 6 Comparison of rAAV Virion Production Efficiency ofPlasmid-Based Accessory Functions

In order to compare the efficiency of rAAV virion production in a hostcell using plasmid-based isolated accessory functions, combinationsthereof, and a single construct containing adenovirus VA RNA, E4 and E2agene regions (the pladeno 1 plasmid, described below) with rAAV virionproduction obtained using adenovirus type-2 (ad-2) infection, thefollowing experiment was carried out.

1. Construction of pladeno 1 and pladeno 1 E1:

The plasmid pladeno 1, containing adenovirus VA RNA, E4 and E2a generegions, was assembled by cloning adenovirus type-5 genes into a custompolylinker that was inserted between the PvuII sites of pBSII s/k−. Amap of the pladeno 1 construct is depicted in FIG. 1. More particularly,a double stranded oligonucleotide polylinker encoding the restrictionenzyme sites SalI-XbaI-EcoRV-SrfI-BamHI

-   (5′-GTCGACAAATCTAGATATCGCCCGGGCGGATCC-3′ (SEQ ID NO:10)) was ligated    to the 2513 bp PvuII vector fragment of pBSII s/k− to provide an    assembly plasmid. The following fragments containing adenovirus    type-5 genes or gene regions were then obtained from the pJM17    plasmid: the 1,724 bp SalI-HinDIII VA RNA-containing fragment    (corresponding to the nucleotides spanning positions about 9,831 to    about 11,555 of the adenovirus type-2 genome); the 5,962 bp    SrfI-BamHI E2a-containing fragment (corresponding to the nucleotides    spanning positions about 21,606 to about 27,568 of the adenovirus    type-2 genome); and the 3,669 bp HphI-HinDIII E4-containing fragment    (corresponding to the nucleotides spanning positions about 32,172 to    about 36,841 of the adenovirus type-2 genome). An XbaI site was    added to the HphI end of the E4-containing fragment by cloning the    3,669 bp HphI-HinDIII fragment into the HpaI site of cloning vector,    and then excising the fragment with XbaI and HinDIII (partial    digestion). The 5,962 E2a-containing fragment was cloned between the    SrfI and BamHI sites of the assembly plasmid, and the 1,724 bp VA    RNA-containing fragment and the modified 3,669 bp E4-containing    fragments were joined by their common HinDIII ends and ligated    between the SalI and XbaI sites of the assembly plasmid to obtain    the pladeno 1 construct.

Referring now to FIG. 2, the pladeno 1 E1 plasmid was assembled asfollows. The 4,102 bp BsrGI-Eco47III fragment (containing the adenovirustype-5 E1a and E1b coding regions) was obtained from the pJM17 plasmid.The subject fragment corresponds to the nucleotides spanning positionsabout 192 to about 4,294 of the adenovirus type-2 genome. The 4,102 bpfragment was blunt-end modified, and then inserted into the HpaI site inthe VA RNA fragment of the pladeno 1 plasmid to obtain the pladeno 1 E1plasmid.

2. rAAV Virion Production Assay:

293 cells were plated in 10-cm tissue culture dishes at 1×10⁶cells/dish, and were cultured at 37° C. to reach approximately 90%confluency over a period of from about 24 to 48 hours prior toco-transfections. All transfections were carried out as described abovein Example 1.

All of the 293 cell cultures were transfected with 5 μg of the plasmidpW1909adhlacZ. Experimental groups of the cultures were alsoco-transfected with various combinations of 5 μg each of the accessoryfunction containing plasmids or control plasmid (pBSII s/k−). After thetransfections, the media was replaced, and the cultures were incubatedat 37° C. for approximately 72 hours. As a comparison, 10 mL of mediumcontaining adenovirus type-2 (moi=1) was added to 293 cells that hadbeen transfected with 5 μg of the plasmid pW1909adhlacZ, and incubatedat 37° C. for approximately 72 hours.

Cells from each experimental group were then collected, media wasremoved by centrifugation (1000×g for 10 min.), and a 1 mL lysate wasproduced using 3 freeze/thaw cycles (alternating between dry ice-ethanoland 37° C. baths). The lysates were made free of debris bycentrifugation (10,000×g for 10 min). rAAV lacZ virion production wasthen assessed using the techniques described in Example 1. The resultsfrom the experiment are depicted below in Table 6.

TABLE 6 Transfected Plasmids AAV Titer† ad-2 (moi = 1) 5 × 10⁹ pladeno 13 × 10⁹ pBSII-E2a, pBSII-E4, 1 × 10⁹ pBSII-VA RNAs pBSII-E2a + E4,pBSII-VA RNAs 5 × 10⁸ pladeno 1, pBSII-E2a 8 × 10⁹ pladeno 1, pBSII-E4 2× 10⁹ pladeno 1, pBSII-VA RNAs 2 × 10⁹ pladeno 1, pBSII-E2a + E4 1 × 10⁹pBSII-E2a 2 × 10⁷ pBSII-E4 <10⁴ pBSII-VA RNAs 3 × 10⁴ pBSII s/k− <10⁴†As determined by the lacZ assay.

As can be seen by the results in Table 6, the pladeno 1 construct iscapable of supporting efficient rAAV virion production (at substantiallythe same level as that obtained using adenovirus type-2 infection). Thecombination of accessory function constructs pBSII-E2a, pBSII-E4 andpBSII-VA RNAs was also able to support efficient rAAV virion productionat levels substantially equivalent to ad-2 infection. The combination ofaccessory function constructs pBSII-E2a+E4 and pBSII-VA RNAs was able tosupport rAAV production (10 fold less than ad-2 infection levels); andthe E2a containing construct (pBSII-E2a) supported rAAV production at alevel approximately 200 fold less than that obtained using ad-2infection.

EXAMPLE 7 Comparison of Large Scale rAAV Virion Production ObtainedUsing Adenoviral-Based or Plasmid-Based Accessory Functions

In order to compare large scale preparations of rAAV virions producedusing either adenovirus type-2 (ad-2) based, or pladeno 1 basedaccessory functions, the following experiment was carried out.

Approximately 10⁹ 293 cells were transfected with an AAV vectorcontaining the human erythropoietin gene using the transfection methoddescribed in Example 1. One preparation was co-transfected with thepladeno 1 construct, the other preparation was infected with adenovirustype-2 as a source of accessory functions.

After a suitable incubation period, cells from each experimental groupwere then collected, growth media was removed by centrifugation (1000×gfor 10 min.), and a lysate was produced using 3 freeze/thaw cycles(alternating between dry ice-ethanol and 37° C. baths). The lysates weremade free of debris by centrifugation (10,000×g for 10 min) to obtain acrude lysate. In order to obtain a purified sample, the crude lysateswere subjected to density gradient centrifugation.

The amount of rAAV genomes produced by each preparation was quantifiedby the dot blot assay described in Example 2. The results of theexperiment are depicted below in Table 7.

TABLE 7 Ad-2 pladeno 1 genomes % recovery genomes % recovery crudelysate 6.5 × 10¹³ 100 1.6 × 10¹⁴ 100 after 5.5 × 10¹³ 85   8 × 10¹³ 50purification

As can be seen from the results in Table 7, the preparation using thepladeno 1-based accessory functions provided a rAAV virion yield thatwas 2.4 fold greater than that obtained from the preparation using theadenovirus type-2 based accessory functions.

EXAMPLE 8 Determination of the Relative Contributions of IndividualAdenoviral Accessory Functions in rAAV Virion Production

In order to determine the relative contributions of the individualadenoviral accessory functions in rAAV virion production, the followingexperiment was carried out. Individual adenoviral accessory functions,either alone, or in combinations, were used to support rAAV virionproduction in host cells. In addition, the effect of substitutingCMV-driven E2a or E4 ORF6 constructs (the p3.3cE2A and p3.3cE4ORF6constructs, described below), for constructs containing the entire E2aand E4 ORF6 regions and driven by homologous promoters (the pBSII-E2aand pBSII-E4 constructs) was assessed.

1. Construction of p3.3cE2A and p3.3cE4ORF6:

The structural genes encoding the adenovirus type-5 E2a 72 kDDNA-binding protein, and the adenovirus type-5 E4 open reading frame 6(ORF6) protein, were each cloned into a CMV driven expression construct,p3.3c, to provide the p3.3cE2A and p3.3E40RF6 plasmid constructs,respectively.

Plasmid p3.3c was constructed as follows. The 2732 bp NotI fragment fromp1.1c, which contains pUC119 vector sequences, was ligated to asynthetic DNA fragment containing the following restriction sites: NotI;MluI; Ecl136II; SacII; BstBI; BssHII; SrfI; BssHII; BglII; SnaBI;BstEII; PmlI; RsrII; and NotI. The sequence of the synthetic DNAfragment is:

-   5′-GCGGCCGCACGCGTGAGCTCCGCGGTTCGAAGCGCGCAAAGCCCCGGGCAAAGCG    CGCAGATCTACGTAGGTAACCACGTGCGGACCGGCGGCCGC-3′ (SEQ ID NO:11). A 653    bp SpeI-SacII fragment including the cytomegalovirus immediate early    (CMV IE) promoter; a 269 bp PCR-produced BstBI-BstBI fragment    encoding the hGH 1st intron that was obtained using the following    primers-   5′-AAAATTCGAACAGGTAAGCGCCCCTTTG-3′ (SEQ ID NO:12) and    3′-AAAATTCGAATCCTGGGGAGAAACCAGAG-5′ (SEQ ID NO:13); and a 213 bp    BamHI-BamHI (blunted) fragment containing the SV40 late    polyadenylation site from pCMV-β (obtained from Clonetech), were    cloned into the Ecl136II, BstBI and SnaBI sites of the synthetic    linker, respectively, to result in the p3.3c expression construct.

Plasmid p3.3cE4ORF6 (ATCC Accession Number 98234) was prepared asfollows. The 1024 bp BglII-SmaI fragment from pBSII-E4, containingsequences encoding the adenovirus type-5 E4 ORF6, was obtained and bluntend-modified. This fragment corresponds to position 33,309 (SmaI site)through position 34,115 (BglII site) of the adenovirus type-2 genome.The modified fragment was then cloned into the SrfI site of p3.3c toprovide the p3.3cE4ORF6 plasmid.

Plasmid p3.3cE2A (ATCC Accession Number 98235) was prepared as follows.The 2467 bp MscI(partial)-BamHI fragment from pBSII-E2a was obtained.This fragment contains adenovirus type-5 E2a coding sequences andcorresponds to positions 24,073 (MscI site) through 21,606 (BamHI site)of the adenovirus type-2 genome. The 2467 bp fragment was subclonedbetween the MscI and BamHI sites of pCITE2A (obtained from Novagene) toprovide the pCITE2AE2A construct. The 1636 bp NcoI(partial)-BsrGIfragment was then excised from pCITE2AE2A. This fragment containssequences encoding the E2a 72 kD protein, and corresponds to positions24,076 (MscI/NcoI site) through 22,440 (BsrGI site) of the adenovirustype-2 genome. The 1636 bp fragment was blunt end-modified, and clonedinto p3.3c to provide the p3.3cE2A plasmid.

2. rAAV Virion Production Assay:

293 cells were plated in 10-cm tissue culture dishes at 2×10⁶cells/dish, and were cultured at 37° C. to reach approximately 90%confluency over a period of about 48 hours prior to co-transfections.All transfections were carried out using the CaPO₄ method with thefollowing combinations of DNAs. All of the 293 cell cultures weretransfected with 5 μg of the plasmid pW620adhlacZ. 5 μg of variousaccessory function constructs (as shown in Table 8 below) were used toprovide a total of 20 μg DNA per dish. For samples receiving less than 4constructs, pBSII DNA was used to bring the total amount of transfectedDNA to 20 μg. After the transfections, the media was replaced, andcultures using adenovirus for accessory functions received adenovirustype-2 at a MOI of 5. All cultures were then incubated at 37° C. forapproximately 72 hours prior to harvest.

Cells from each dish were then collected, media was removed bycentrifugation (1000×g for 10 min.), and a 1 mL lysate was producedusing 3 freeze/thaw cycles (alternating between dry ice-ethanol and 37°C. baths). The lysates were made free of debris by centrifugation(10,000×g for 10 min). rAAV lacZ virion production was then assessedusing the techniques described in Example 1. The lacZ titering was donein the presence of adenovirus. All samples were assayed in duplicate.The results from the experiment are depicted below in Table 8.

TABLE 8 Adenoviral Accessory Functions AAV Titer† ad-2 (moi = 5) 4 × 10⁸pBSII-E2a, pBSII-E4, pBSII-VA RNAs 6 × 10⁸ pladeno 1 8 × 10⁸ pBSII-E2a 8× 10⁶ PBSII-E4 <10⁴ pBSII-VA RNAs 5 × 10⁴ pBSII-E4, pBSII-VA RNAs 1 ×10⁵ pBSII-E2a, pBSII-VA RNAs 6 × 10⁷ pBSII-E2a, pBSII-E4 1 × 10⁸p3.3cE2A, pBSII-E4, pBSII-VA RNAs 4 × 10⁸ pBSII-E2a, p3.3cE4ORF6,pBSII-VA RNAs 3 × 10⁸ p3.3cE2A, p3.3cE4ORF6, pBSII-VA RNAs 4 × 10⁸ †Asdetermined by the lacZ assay.

As can be seen by the results depicted in Table 8, the adenoviral E2a,E4 and VA RNA regions are all necessary to provide efficient rAAV virionproduction (at levels substantially equivalent to those obtained usingadenovirus infection to provide the accessory functions). However, nosingle one of those regions is absolutely required for rAAV virionproduction.

The E2a and E4 regions that are subcloned into the pBSII-E2a andpBSII-E4 constructs contain several open reading frames (ORFs) inaddition to the ORFs for the 72 kD E2a DNA binding protein and the E4ORF6-protein. By substituting the CMV-driven p3.3cE2A and p3.3cE4ORF6constructs for the pBSII-E2a and pBSII-E4 constructs in theabove-described study, it has now been established that the E2a 72 kDDNA-binding protein and the E4 ORF6 protein are capable of providingfull E2a or E4 accessory function in the absence of all other openreading frames from their respective coding regions.

EXAMPLE 9 Reconstruction of the Pladeno 1 Plasmid and Comparison of rAAVVirion Production Efficiency

The pladeno 1 plasmid was reconstructed using purified adenovirus type-2DNA as a source of the adenoviral genes in place of the pJM17-derivedadenovirus genes that were used in the construction of pladeno 1. Thereconstructed plasmid, termed pladeno 5, is described in detail below.This reconstruction was carried out to reduce the overall size of theplasmid. Furthermore, the reconstructed plasmid (pladeno 5) does notencode the adenovirus protease, whereas pladeno 1 does.

1. Construction of Pladeno 5:

The pladeno 5 plasmid was constructed as follows. DNA fragments encodingthe E2a, E4 and VA RNA regions isolated from purified adenovirus type-2DNA (obtained from Gibco/BRL) were ligated into a plasmid calledpAmpscript. The pAmpscript plasmid was assembled as follows:oligonucleotide-directed mutagenesis was used to eliminate a 623 bpregion including the polylinker and alpha complementation expressioncassette from pBSII s/k+ (obtained from Stratagene), and to replace itwith an EcoRV site. The sequence of the mutagenic oligo used on theoligonucleotide-directed mutagenesis was

-   5′-CCGCTACAGGGCGCGATATCAGCTCACTCAA-3′ (SEQ ID NO:14). A polylinker    (containing the following restriction sites: BamHI; KpnI; Srfl;    XbaI; ClaI; Bst1107I; SalI; PmeI; and NdeI) was synthesized and    inserted into the EcoRV site created above such that the BamHI side    of the linker was proximal to the f1 origin in the modified plasmid    to provide the pAmpscript plasmid. The sequence of the polylinker    was-   5′-GGATCCGGTACCGCCCGGGCTCTAGAATCGATGTATACGTCGACGTTTAAACCAT ATG-3′    (SEQ ID NO:15).

DNA fragments comprising the adenovirus type-2 E2a and VA RNA sequenceswere cloned directly into pAmpscript. In particular, a 5962 bpSrfI-KpnI(partial) fragment containing the E2a region was cloned betweenthe SrfI and KpnI sites of pAmpscript. The 5962 bp fragment comprisesbase pairs 21,606-27,568 of the adenovirus type-2 genome. A 732 bpEcoRV-SacII(blunted) fragment containing the VA RNAs was cloned into theBst1107I site of pAmpscript. The 732 bp fragment is equivalent to basepairs 10,423-11,155 of the adenovirus type-2 genome.

The DNA comprising the adenovirus type-2 E4 sequences had to be modifiedbefore it could be inserted into the pAmpscript polylinker.Specifically, PCR mutagenesis was used to replace the E4 proximal,adenoviral terminal repeat with a SrfI site. The location of this SrfIsite is equivalent to base pairs 35,836-35,844 of the adenovirus type-2genome. The sequences of the oligonucleotides used in the mutagenesiswere:

-   5′-AGAGGCCCGGGCGTTTTAGGGCGGAGTAACTTGC-3′ (SEQ ID NO:16); and    5′-ACATACCCGCAGGCGTAGAGAC-3′ (SEQ ID NO:17). A 3,192 bp E4 fragment,    produced by cleaving the above-described modified E4 gene with SrfI    and SpeI, was ligated between the SrfI and XbaI sites of pAmpscript    which already contained the E2a and VA RNA sequences to result in    the pladeno 5 plasmid. The 3,192 bp fragment is equivalent to base    pairs 32,644-35,836 of the adenovirus type-2 genome.    2. rAAV Virion Production Assay:

293 cells were plated in 10-cm tissue culture dishes at 1×10⁶cells/dish, and were cultured at 37° C. to reach approximately 90%confluency over a period of from about 24 to 48 hours prior toco-transfections. All transfections were carried out as described abovein Example 1.

The 293 cell cultures were transfected with 5 μg of the pVlacZ vectorplasmid, 5 μg of plasmid pW1909, and 5 μg of either pladeno 1 or pladeno5. After the transfections, the media was replaced, and the cultureswere incubated at 37° C. for approximately 72 hours before harvest.

Cells from each experimental group were then collected, media wasremoved by centrifugation (1000×g for 10 min.), and a 1 mL lysate wasproduced using 3 freeze/thaw cycles (alternating between dry ice-ethanoland 37° C. baths). The lysates were made free of debris bycentrifugation (10,000×g for 10 min). rAAV lacZ virion production wasthen assessed using the techniques described in Example 1. The resultsfrom the experiment are depicted below in Table 9

TABLE 9 Accessory Function Vector rAAV Titer† pladeno 1 2.2 × 10⁹pladeno 5 5.5 × 10⁹ †As determined by the lacZ assay.

As can be seen by the results reported in Table 9, greater rAAV virionproduction efficiency was seen when using pladeno 5 (pladeno 5production yielded 2.5 fold more rAAV virions than pladeno 1production).

Accordingly, novel accessory functions capable of supporting efficientrecombinant AAV virion production have been described. Althoughpreferred embodiments of the subject invention have been described insome detail, it is understood that obvious variations can be madewithout departing from the spirit and the scope of the invention asdefined by the appended claims.

Deposits of Strains Useful in Practicing the Invention

A deposit of biologically pure cultures of the following strains wasmade with the American Type Culture Collection, 12301 Parklawn Drive,Rockville, Md., under the provisions of the Budapest Treaty. Theaccession number indicated was assigned after successful viabilitytesting, and the requisite fees were paid. Access to said cultures willbe available during pendency of the patent application to one determinedby the Commissioner to be entitled thereto under 37 CFR 1.14 and 35 USC122. Upon the granting of a patent-in this application, all restrictionsimposed by the depositor on the availability to the public of thedeposited biological material will be irrevocably removed with thefollowing one exception, as specified in 37 C.F.R. §1.808(b), saidexception being that depositor reserves the right to contract with thedepository to require that samples of the deposited material befurnished only if a request for a sample, during the term of the issuedpatent, meets any one, or all, of the following three conditions: (1)the request is in writing or other tangible form and dated; and/or (2)the request contains the name and address of the requesting party andthe accession number of the deposit; and/or (3) the request iscommunicated in writing by the depository to the depositor along withthe date on which the sample was furnished and the name and address ofthe party to whom the sample was furnished. Moreover, the designateddeposits will be maintained for a period of thirty (30) years from thedate of deposit, or for five (5) years after the last request for thedeposit; or for the enforceable life of the U.S. patent, whichever islonger. Should a culture become nonviable or be inadvertently destroyed,or, in the case of plasmid-containing strains, lose its plasmid, it willbe replaced with a viable culture(s) of the same-taxonomic description.

These deposits are provided merely as a convenience to those of skill inthe art, and are not an admission that a deposit is required. Thenucleic acid sequences of these plasmids, as well as the amino sequencesof the polypeptides encoded thereby, are controlling in the event of anyconflict with the description herein. A license may be required to make,use, or sell the deposited materials, and no such license is herebygranted.

Strain Deposit Date ATCC No. pBSII-VA RNAs Oct. 30, 1996 98233p3.3cE4ORF6 Oct. 30, 1996 98234 p3.3cE2A Oct. 30, 1996 98235 pGN1909Jul. 20, 1995 69871

1. A nucleic acid molecule which provides one or more accessoryfunctions for supporting recombinant AAV (rAAV) virion production in asuitable host cell and that lacks at least one adenoviral late generegion, said molecule comprising a nucleotide sequence selected from thegroup consisting of (i) a sequence that provides adenovirus VA RNAs,(ii) an adenovirus E4 ORF6 coding region, (iii) an adenovirus E2a 72 kDcoding region, and any combination of nucleotide sequences (i), (ii) and(iii).
 2. An accessory function vector comprising the nucleic acidmolecule of claim
 1. 3. The accessory function vector of claim 2,wherein said vector is a plasmid.
 4. The accessory function vector ofclaim 3 further comprising at least one heterologous promoter regionoperably linked to said nucleotide sequence.
 5. The accessory functionvector of claim 4 wherein the at least one heterologous promoter regioncomprises a nucleotide sequence substantially homologous to thecytomegalovirus (CMV) immediate early promoter region.
 6. A nucleic acidmolecule which provides accessory functions for supporting efficientrecombinant AAV (rAAV) virion production in a suitable host cell andthat lacks at least one adenoviral late gene region.
 7. The nucleic acidmolecule of claim 6, wherein said nucleic acid molecule lacks adenoviralearly gene regions 2b and
 3. 8. The nucleic acid molecule of claim 6,which provides accessory functions capable of supporting efficient rAAVvirion production in a human 293 host cell.
 9. The nucleic acid moleculeof claim 8, comprising one or more nucleotide sequences derived from anadenovirus type-2 or type-5 genome.
 10. The nucleic acid molecule ofclaim 9, comprising: a first nucleotide sequence that provides anadenovirus VA RNA; a second nucleotide sequence comprising an adenovirusE4 coding region containing the ORF 6; and a third nucleotide sequencecomprising an adenovirus E2a coding region.
 11. The nucleic acidmolecule of claim 6, wherein the nucleic acid molecule providesaccessory functions capable of supporting efficient recombinant AAV(rAAV) virion production in a suitable host cell that is not infectableby adenovirus or is not capable of supporting adenovirus replication.12. The nucleic acid molecule of claim 11, comprising one or morenucleotide sequences derived from an adenovirus type-2 or type-5 genome.13. The nucleic acid molecule of claim 12, comprising: a firstnucleotide sequence that provides an adenovirus VA RNA; a secondnucleotide sequence comprising an adenovirus E4 coding region containingthe ORF 6; a third nucleotide sequence comprising an adenovirus E2acoding region; and a fourth nucleotide sequence comprising theadenovirus E1a and E1b coding regions.
 14. An accessory function vectorcomprising the nucleic acid molecule of claim
 6. 15. The accessoryfunction vector of claim 14, wherein said vector is a plasmid.
 16. Anaccessory function vector comprising the nucleic acid molecule of claim11.
 17. The accessory function vector of claim 16, wherein said vectoris a plasmid.
 18. The accessory function vector of claim 15 furthercomprising a selectable genetic marker.
 19. The accessory functionvector of claim 18, wherein the selectable genetic marker comprises anantibiotic resistance gene.
 20. The accessory function vector of claim15 further comprising at least one heterologous promoter region.
 21. Theaccessory function vector of claim 20, wherein the at least oneheterologous promoter region comprises a nucleotide sequencesubstantially homologous to the cytomegalovirus (CMV) immediate earlypromoter region.
 22. A host cell that has been transfected with theaccessory function vector of claim
 2. 23. A host cell that has beentransfected with the accessory function vector of claim
 14. 24. A hostcell that has been transfected with the accessory function vector ofclaim
 16. 25. A cell capable of producing recombinant AAV (rAAV) virionswhen transfected with an AAV vector, said cell comprising the host cellof claim 23 co-transfected with an AAV helper construct that is capableof being expressed in said cell to provide AAV helper functions.
 26. Amethod of producing recombinant AAV (rAAV) virions, comprising: (a)introducing an AAV vector into a suitable host cell; (b) introducing anAAV helper construct into the host cell, said helper constructcomprising AAV coding regions that are expressed in the host cell tocomplement AAV helper functions missing from said AAV vector; (c)introducing the accessory function vector of claim 14 into the hostcell, said accessory function vector providing accessory functions forsupporting efficient rAAV virion production in the host cell; and (d)culturing the host cell to produce rAAV virions.
 27. A method ofproducing recombinant AAV (rAAV) virions in a host cell that is notinfectable by adenovirus or is not capable of supporting adenovirusreplication, comprising: (a) introducing an AAV vector into the hostcell; (b) introducing an AAV helper construct into the host cell, saidhelper construct comprising AAV coding regions that are expressed in thehost cell to complement AAV helper functions missing from said AAVvector; (c) introducing the accessory function vector of claim 16 intothe host cell, said accessory function vector providing accessoryfunctions for supporting efficient rAAV virion production in the hostcell; and (d) culturing the host cell to produce rAAV virions.